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THE UNIVERSITY 
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MAY 23 1944 


OCT 29 1959 
JAN 13 19 | 


17625-S 


LECTURES ON 


NUTRITION 


A SERIES OF LECTURES GIVEN AT THE MAYO 

FOUNDATION AND THE UNIVERSITIES OF WIS- 

CONSIN, MINNESOTA, NEBRASKA, IOWA, AND 
WASHINGTON (ST. LOUIS) 


1924-1925 


ILLUSTRATED 


PHILADELPHIA AND LONDON 


W. B. SAUNDERS COMPANY 


Copyright, 1925, by W. B. Saunders Company 


MADE IN U. 8. A. 


PRESS OF 
W. B, SAUNDERS COMPANY 
PHILADELPHIA 


¢ INTRODUCTION 


The lectures on nutrition herewith presented to the public 
were given during the year 1924-1925 under the auspices of 
the Mayo Foundation and the local chapter of Sigma Xi at 
Rochester, Minnesota; the Medical School of the University 
of Wisconsin at Madison, Wisconsin; the Graduate School of 
the University of Minnesota at Minneapolis, Minnesota; the 
Medical School of the University of Nebraska at Omaha, 
Nebraska; the Medical School of Washington University at 
St. Louis, Missouri; the Graduate School of the University 
of Iowa at Iowa City, Iowa, and the Des Moines Academy 

_ of Medicine at Des Moines, Iowa. 

% The lectures include a large portion of the recent research 

M ‘ work in the field of nutrition. The lecturers were the persons 
who had conducted or been responsible in large measure for 
ue the several researches. The volume therefore is a statement 
«by competent authorities of our present-day knowledge of 
is most of the important problems of nutrition. While the 
Adectures do not attempt to cover in detail the entire subject 
\.. of nutrition, they do contain a large body of fresh information 
4 on the subject which it is believed will prove of live interest 
La to the public. 


sR Louis B. Witson, M. D. 
* Director 
ihe ROCHESTER, MINNESOTA. The Mayo Foundation for 
November, 1925. Medical Education and Research. 
13 


bO23 774 


Pets, 5 pis f 


ees wt 


CONTENTS 


PAGE 

THE MEASUREMENT AND SIGNIFICANCE OF BASAL METABOLISM.... 17 
Francis Gano Benedict, Director Nutrition Laboratory, 
Carnegie Institution of Washington, Boston, Massachusetts. 

PROBCEMSAOFS VIETA BOLISMie tern ue eesti. eae ae a tte ne a ues 59 


Graham Lusk, Scientific Director Russell Sage Institute of 
Pathology, Professor of Physiology, Cornell University, New 
York. 


THE PROPORTIONS IN WHICH PROTEIN, FAT, AND CARBOHYDRATE ARE 
METABOLIZED TIN SLISEASE ot ote ctene ate ay 6 sete te 77 
Eugene Floyd DuBois, Medical Director Russell Sage In- 
stitute of Pathology, Associate Professor of Medicine, Cornell 
University, New York. 


Muscutar ACTIVITY AND CARBOHYDRATE METABOLISM........... 109 
Archibald Vivian Hill, Professor of Physiology, University 
College, London, England. 


OuR PRESENT KNOWLEDGE OF THE VITAMINS............--20005- 137 
Elmer Verner McCollum, Professor and Head of Depart- 
ment of Chemical Hygiene, School of Hygiene and Public 

. Health, Johns Hopkins University, Baltimore, Maryland. 


THE RELATIONS BETWEEN FERTILITY AND NUTRITION............ 209 
Herbert McLean Evans, Professor of Anatomy, University 
of California, Berkeley, California. 


{5 


LECTURES ON NUTRITION 


THE MEASUREMENT AND SIGNIFICANCE OF BASAL 
METABOLISM 


FRANCIS GANO BENEDICT 


As a result of the war the American people have become 


familiar with the word “calorie,” 


and have begun to think 
of foods in terms of heat units. The word “vitamin” (that 
elusive but necessary food-accessory factor) has also appeared 
in the vocabulary pertaining to nutrition, and now the word 
“metabolism” bids fair to be firmly established therein. A 
calorie is a heat unit easily definable, if not easily compre- 
hensible. Vitamins are things as yet unseen or at least 
seen but evanescently. Metabolism is a deep-seated life 
process, the products of which are in part visible, such as 
fat deposits in the body, and in large part invisible, as in the 
products of combustion. 


DEFINITION OF METABOLISM 


In spite of its complexity of meaning, the word ‘‘metab- 
olism” is now in current use. Studies and measurements of 
metabolism are being made, and we hope that this process 
is being better understood. In its full meaning metabolism 


indicates the changes, constructive and destructive, which 
2 17 


18 LECTURES ON NUTRITION 


take place in a living organism. In plants, obviously, the 
process is chiefly a constructive one, that is, the synthesizing 
of the carbohydrate of the cellulose and starch cells from the 
carbon dioxid of the air and water. In the animal those forces 
in metabolism producing growth represent the constructive 
phase, while the ordinary disintegration during the life proc- 
esses represents the destructive phase. 

The word “metabolism” formerly had a wide significance, 
for it included all transformations of matter and energy in 


BODY MATERIAL 


ey 4%, 
Y, 4 
S) o 
® Q 
% oy 
Y 


FOOD END PRODUCTS 


(CO,,H,0, UREA, ETC) 


METABOLISM 


Bigs 1: 


the body, that is, the processes of synthesis and of analysis. 
A simple chart helps to visualize these processes. 

From this chart it is obvious that the constructive forces 
by which food materials are used to build up the body rep- 
resent the anabolic changes, while the ordinary life processes, 
particularly when food is not given, are the katabolic processes. 
It is extremely difficult to make any real quantitative esti- 
mates of the processes of anabolism. By careful analyses of 
both income and outgo it is possible to find out if there has 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 19 


been at any time a preponderance of anabolic changes, that 
is, if there has been a gain of chemical elements to the or- 
ganism. ‘This gain, of course, takes place chiefly during the 
period of growth. But such a measurement must of neces- 
sity in most cases be based simply upon a differential method. 
If the intake is larger than the output, the difference is con- 
sidered to have been retained by the body. But to determine 
the exact course of any given food molecule in its transfor- 
mation into body substance, and particularly to attempt to 
localize such a transformation, is beyond present-day tech- 
nic. Anabolic processes have, therefore, been but little studied. 
If in a long period of excess feeding with carbohydrates a 
pig or a goose is fattened, we properly speak of a transfor- 
mation of carbohydrate to fat. With human beings, a great 
many women and not a few men over forty know that such 
transformations are only too possible. But quantitative 
measures of such processes are practically unknown. 

Strictly speaking, the transformations of any compounds, 
organic or inorganic, in the body are processes of metab- 
olism, and since the nitrogenous transformations can all 
be measured by determining the nitrogen of income and the 
nitrogen of output (that is, the nitrogen in urine and feces) 
we often speak of “nitrogenous metabolism.” The salts of 
calcium and magnesium and the compounds of phosphorus 
and sulphur are now regularly studied and reports are pub- 
lished on the metabolism of these elements. Yet the new 
trend, certainly among the laity and those whose friends 
have been in metabolism clinics, is to limit the meaning of 
the word to the metabolism of energy, especially as represented 


20 LECTURES ON NUTRITION 


by the gaseous exchange. Hence the application of the word 
“metabolism” is being more and more restricted to gaseous 
metabolism. 

While the current meaning of metabolism excludes con- 
structive or synthetic processes, since there is little or no 
heat involved in such processes, and excludes fermentations 
which involve but little heat, or enzyme action with its slight 
heat production, it does include those extensive breaking- 
down processes or oxidations accompanied by the production 
of large quantities of heat. In clinical medicine, the term 
“metabolism” and especially “basal metabolism” is today 
for the most part restricted to basal katabolism or the break- 
ing down of body materials (chiefly fat and carbohydrate) 
by the processes of oxidation, whereby substantial amounts 


of heat are produced. 


FACTORS INVOLVED IN METABOLISM 


Clearly recognizing, therefore, that by metabolism we 
mean basal katabolism and that we are interested in the 
katabolic and not the anabolic processes, we may proceed to 


consider the factors involved in metabolism. 


INGESTION OF FOOD 


When food is eaten, both processes, anabolism and kata- 
bolism, are at work. In studying the transformations of 
matter and energy, however (which are in large part, al- 
though not exclusively katabolic), it is highly desirable and 
fortunately very practical to minimize, if not altogether to 
rule out, anabolism by completely withholding food. Even 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 21 


with food withdrawal, when katabolic processes prepon- 
derate, there may be anabolic changes, for there are well- 
known instances of fish which not only live for a long time 
without food, but actually undergo great anatomic changes 
as a result of anabolic processes when fasting. The salmon 


TOS cee 


enters the river from the sea with well-developed muscles, | 


and proceeds up the river to the spawning grounds. While | 


in transit no food is taken and the sexual organs become 
greatly enlarged by the transportation of nitrogenous ma- 
terial from the muscles, which become correspondingly re- 
duced. 

With all living organisms in the early period following food 
withdrawal, while there is still unabsorbed and undigested 
food in the alimentary tract, the anabolism may still be going 
on, indeed until the content of the alimentary tract no longer 
yields material to the blood stream. In the case of man, with 


small contents in the digestive tract, this period is relatively 


short, at the most twenty-four hours. A ruminant, such 
as the ox, however, has a large intestinal fill, amounting in 
a well-fed ox to one-fifth of the total body weight; and this 


mass furnishes at least a portion of the energy for life for. 


from four to five days after withdrawal of food. 
At the New York Zodlogical Park Mr. E. L. Fox of the 


Nutrition Laboratory staff has found that with large snakes | 
the period of digestion may easily be prolonged a week or | 


more by keeping them at a temperature below 20° C. 

In any consideration of basal metabolism, therefore, it is 
most important to take into consideration the influence of 
food, for the ingestion of food causes an increase in metab- 


22 LECTURES ON NUTRITION 


olism; and although anabolic processes predominate, there 
is always a stimulus to katabolism and increased heat pro- 
duction. 

DIFFERENCES IN INDIVIDUALS 

That the ingestion of food increases circulation and gives 
a feeling of warmth is noted by everyone. There, is there- 
fore, no question but what there is increased functional ac- 
tivity as a result of the ingestion of food. But not all factors 
which might affect metabolism are so easily recognized. 
Indeed, the heat production of human beings varies greatly 
with different individuals, and in the same organism the 
heat output is extremely variable. It is important, there- 
fore, to attempt to catalogue those factors known to in- 
fluence metabolism, and we may first consider the differences 
between different organisms, such as between men and 
boys, or women and girls. 

It is obvious that a large organism will produce more 
heat than a smaller organism, and yet we are immediately 
confronted with the problem of the units of measurement 
to be employed in the comparison. Size may be indicated 
by at least three factors, weight, height, or the more com- 
monly used surface area. The difficulty of isolating the 
effect of each of these three factors on metabolism can hardly 
be overemphasized. An analysis of the factors going to make 
up weight alone shows that we have to deal with skeleton, 
muscular mass, and fat. The oxidative processes are in 
large part centered in the muscular ‘mass, and we have every 
reason to believe that body fat is more or less inert, so far 


as oxidations are concerned. Thus, a weight made up in 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 23 


large part of body fat would be, so to speak, diluted with 
an inert material. 

The exact valuation of the factors of weight, height, and 
surface area is best made by a most careful biometric anal- 
ysis of a large number of metabolism measurements with in- 
dividuals of varying sizes. From this biometric analysis" 
it seems clearly established that weight and height are both 
independent factors. With regard to surface area a great 
deal of discussion has been raised. In general it is perfectly 
proper to state that those persons having larger surface areas 
will have a larger heat output. Indeed, it is stoutly main- 
tained by most physiologists that the heat production per 
square meter of surface area is constant for all warm-blooded 
animals, whether we are dealing with a man, horse, dog, or 
mouse. The Nutrition Laboratory’s experience does not 
support this view. 

Entirely aside from these anatomical factors of weight, 
height, and surface area, it has been definitely proved that 
age, independent of size, is also a factor which influences 
metabolism. ‘Thus, the older the person, the less the amount 
of heat produced. In addition, sex has a definite influence, 
for women and girls have a considerably lower heat produc- 


tion than men and boys of the same size. 


MUSCULAR ACTIVITY 


Aside from the foregoing factors affecting metabolism in 
different organisms, there are others which affect metab- 
olism even in the same organism where weight, height, 


age, sex, surface area, and muscular mass remain constant. 


24 LECTURES ON NUTRITION 


Consequently these factors must be recognized in any con- 
sideration of basal metabolism. 

Muscular activity is the most important of these, for the 
smallest muscular motions increase heat production. Severe 
muscular work has a very great influence. Thus a man work- 
ing to the limit of human endurance can for some time in- 
crease his heat production tenfold. ‘The pronounced after- 
effects of work, especially of severe work, should also be con-’ 
sidered. The work of walking up stairs, for example, has an 
after-effect which increases the metabolism for some time, 
and it is not until the person has lain down for at least one- 


half hour that this after-effect disappears. 


ENVIRONMENTAL TEMPERATURE 


Environmental temperature under ordinary conditions of 
laboratory measurement does not play a great réle, but ex- 
periments in which there have been large temperature changes, 
particularly experiments with animals, have shown that the 
lower the environmental temperature the greater the heat 
production. 

OTHER FACTORS 

Psychic disturbances are accompanied by increased met- 
abolism. Hence calm, repose, and quiet are essential. 

Any fever is accompanied by increased heat production, 
for as the cells are warmed there is greater metabolic activity. 

Sleep lowers metabolism, and is as yet a factor too little 
reckoned with. 

State of nutrition is another factor to be considered. Changes 
in weight may be due, in the child, to natural processes of 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 25 


growth, that is, the formation of flesh, fat, and skeleton. 
Changes in weight may also be due to loss of flesh or dis- 
turbances of balance between flesh and fat. A well-nourished 
person has a higher metabolism than one who is poorly 
nourished. A_loss of weight of 10 per cent may be accom- 
panied by a decrease in metabolism of 20 per cent. 


HEAT PRODUCTION AND HEAT LOSS 


Thus we see that the fires of life (as recorded by the meas- 
urements of the calorimeter or by the analyses of the exhaled 


Fig. 2.—Skin temperatures at different parts of the body, under clothing. 


air) burn with varying intensity and are subject to many 
influences. The measurement of the intensity of combustion 
by direct calorimetry deals only with heat output or heat 
given off by the body, and not with heat production. Heat 
production and heat loss are two very different things. Heat 
production is relatively stable under conditions of normal 


26 LECTURES ON NUTRITION 


Front 


= +—_— + —___4 _»__4 __xzv ae 


eee 
Chest | Waist [Thigh [Shin | 
N ipple Groin Knee Ankle 


Back 


24.7 = ——— = 


= —-— E * —t + + = 


l | 
Shays Buttock | Calf 
Waist Knee Ankle 


Fig. 3.—Photographic records from a string galvanometer for skin 
temperatures at different parts of the nude body following a one-minute 
exposure to an environmental temperature of 14.6° C. The thermal junc- 
tion was moved at constant tempo from shoulder to ankle. Time registered 


at bottom of each curve is in two-second intervals. 


repose, that is, there are no sudden changes unless one has 


muscular activity to deal with. 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 27 


Heat Joss to the environment is a very labile thing. The 
warm, living body has a normal internal temperature of 37° C. 
and a peripheral or skin temperature under the clothing, at 
different parts of the body, varying from 28° to 35° C. The 
environmental temperature may be very low, and the heat 
loss to the environment will naturally alter with the varying 
temperature gradients between the skin temperature and the 
environmental temperature. The flow of blood to the skin 
will alter the skin temperature and hence the rate of heat loss. 
The taking off of protective coverings such as gloves, hat, | 
coat, or shawl, or the opening of an overcoat will instantly 
accelerate heat loss. , 

The temperature of the skin is variable, depending in large 
part on the rate of heat loss and much less on the heat pro- 
duction. Even under the clothing of .a comfortably dressed 
person the skin temperature is by no means uniform (Fig. 2). 

If the body is exposed, nude, to a room air of varying 
temperature, there are profound alterations in heat loss, as 
shown by the fall in skin temperature. 

In Figure 3 the curve was made by connecting the thermo- 
junction with an Einthoven galvanometer and drawing the 
junction slowly down over the right mammillary line. Before 
exposure the temperature of the trunk is more nearly uni- 
form, and the effect of exposure of but one minute is clearly 
seen. 

DIRECT AND INDIRECT CALORIMETRY 

The course of the heat production under these conditions 
can be studied only by indirect calorimetry, that is, by 
measuring the oxygen intake and the carbon-dioxide output. 


28 LECTURES ON NUTRITION 


Innumerable tests show that with prolonged exposure there 
is an increase in the heat production, but it is not at all of 
the order of magnitude of the heat loss. Thus, with exposure 
to cold there is a great loss of heat and a slow, small increase 
in heat production. The body as a whole, therefore, has lost 
previously stored heat. ‘This is proved by placing a ther- 
mometer between the clasped hands, or between the crossed 
arms, or the crossed legs, when a low temperature is found 
not only on the skin, but for the deep tissues, which have 
lost previously stored heat. This loss of heat (which could 
be measured in a calorimeter) is made up probably only in 
small part by an increased heat production. 

Basal metabolism is not concerned primarily with labile 
heat loss, whether by radiation from the skin or vaporiza- 
tion of water from the lungs and skin; it is concerned with 
heat production. Bo eae 

Practically, therefore, basal metabolism cannot, in human 
beings, be advantageously determined by direct calorimetry, 
unless the experimental periods are long, and many persons 
find it difficult to keep still long enough. Fortunately heat 
production is easily measured by indirect calorimetry, that 
is, by the measurement of oxygen consumption and carbon- 
dioxide production. In ten minutes, by modern methods, a | 
|measurement may be made and the result calculated. 


REPRODUCIBLE EXPERIMENTAL CONDITIONS 


The methods of indirect calorimetry (and they are legion) 
are satisfactory only if strictest attention is paid to the funda- 


mental, standard conditions under which such measurements 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 29 


should be made. Physiologists, knowing the profound in- 
fluence of certain factors on the heat production, have stip- 
ulated that for such measurements the subject should be in 
complete muscular repose and in the post-absorptive condi- 
tion, that is, at least twelve hours must have elapsed since 
the last meal. aaa Gh i 

The modern scientist must furthermore anticipate, if pos- 
sible, the existence of other factors, the importance of which 
will soon be recognized. Such foresight is essential, if the 
results of present tests are not to be rendered valueless in a 
decade because they were obtained without consideration of 
the influence of factors as yet perhaps unlisted. 


MUSCULAR WORK OR REPOSE 


The factor which most profoundly and most promptly 
affects basal metabolism is muscular work. After severe 
muscular work metabolism may be increased 1,000 per cent 
_within two minutes. eek ent els 

Theoretically, every muscular movement is accompanied 
by heat production. Hence, theoretically at least, there 
should be complete muscular repose in all metabolism ex- 
periments. Certainly obvious voluntary muscular movements 
should be repressed. But the question arises as to how ab- 
solute this repression should be. So strong has this insistence 
on muscular repose become that conscientious observers 
reject experiments contaminated by visible activity, and 
so rigidly enforce inactivity that the muscular restraint 
becomes positively painful, if not unbearable, to many 
subjects. 


30 LECTURES ON NUTRITION 


MINOR MUSCULAR MOVEMENTS 

Lefévre,” of Paris, has criticized the emphasis which has 
been laid on the importance of muscular repose. By cal- 
culating the actual foot pounds required to raise the hand to 
the head, he shows that such a movement involves an in- 
significant amount of muscular work. To put the matter to 
test, a series of experiments were made by Mrs. Cornelia 
Golay Benedict and myself in the Nutrition Laboratory. 
With an especially quiet and well-trained subject the basal 
metabolism was first determined in complete repose, and then 
during a simple arm movement, such as raising the hand to 
the forehead every four seconds, that is, fifteen ‘‘silent salutes” 
per minute. The actual increase in metabolism (Table 1) 
proved to be but slight (as Lefévre predicted), 1.5 c.c. of 
oxygen for each movement of the hand. 

If such a movement of the hand once in ten minutes or 
even once a minute is negligible, one can fairly ask how a 
gross movement of the legs should be considered. To test 
this point the subject, while lying quietly, was asked to cross 
and uncross the legs once every twenty seconds. ‘This, as 
well as the raising of the hand, was done to the beat of a 
metronome. ‘The observations indicated that one such leg 
movement a minute would noticeably raise the basal metab- 
olism. One movement in ten minutes, however, would be 
without significance. 

While, therefore, isolated minor muscular movements, such 
as moving the hand to the head and back, have no influence 
on basal metabolism, movement of the legs must be pro- 
scribed. If the stringent rules of repose are to be relaxed 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 31 


TABLE. st 


INFLUENCE ON THE OXYGEN CONSUMPTION OF SMALL MUSCULAR 
MOVEMENTS OF ARMS AND LEGS 


Subject A. Subject B. 
i ead Oxygen oe 4 ore Oxygen 
Date and condition, : consump- ate and condition, A consump- 
1924. Period. tion per Period. or. ne 
minute, c.c. ‘| minute, c.c. 

January 3: February 29: 

IBasaleae «atest. coe I 193 Basalananterwicier, ote I 256 

Basalt mutacin sce iit 188 Basal sere cere cs JE 255 

Arm movement.... III 218 (Basal aire e, ye III 253 

Basa leer ceo eee IV 195 Arm movement... IV 280 
January 7 March 1 

Basa lees seve teeters I 200 IBASA LEIA ee ieaaees Bustice I 257 

‘Basal he eee II 196 Bacal ae se eee te II 255 

Arm movement.... III 210 ae aSal Ce ove wate cs mye Ill 244 

Basaleeten fetes IV 193 Arm movement.... IV 268 

Leg movement..... V 222 Leg movement.... V 285 
January 9: ; 

Basal Steen strate ore I 203 

Basalarim eee II 189 

Arm movement.... Il 224 

Leg movement..... IV 209 

Basal ees tionta cen V 189 
January 16: 

Basaien vaso eae tine I 202 

Basal tae S fishes II 195 

Arm movement... . sail 215 

Leg movement..... IV 225 

iBasale asec ea. | V 200 


*TIn basal periods subjects were lying, clothed, and covered with light blanket. In the arm 
movements the hand was raised to the forehead every four seconds. In the leg movements the 
feet were crossed every twenty seconds. 


a particle (and it is certain that with some persons they should 
be somewhat relaxed) one always runs the danger of letting 
the bars down too much. A good rule is to insist on repose, 
complete if possible, but not such as to result in tension or 
distress, for moderate repose is far better than a tense or 
cramped position. Muscular activity greater than slight, 
visible muscular actions justifies ruling out the period of 
measurement, and it should be ruled out before the analysis 
or computation is made. 


32 LECTURES ON NUTRITION 
‘ 


INFLUENCE OF BODY POSITION 

Since the lying position is commonly believed to be more 
conducive to complete relaxation than the sitting position, 
metabolism measurements are usually made with the subject 
lying. A study has recently been carried out by Mrs. Bene- 
dict and myself on the influence of posture on metabolism,* 
and we have added our data to the somewhat extended earlier 
data. With a trained artist’s model the metabolism was 
determined in the three different positions. The comparison 


between lying and sitting is shown in Table 2. 


TABLE 2 


COMPARISON OF THE OXYGEN CONSUMPTION PER MINUTE IN THE LYING 
AND SITTING POSITIONS. 


Date. Lying (awake), Sitting, 

(ery ey (OM ott 
December! 11920 2 250 nie ee eee 180 191 
December3 21920 sone te eee eee 173 186 
December 167192005 7 Fuge einen a: 180 188 
Decemberal/, 19200 eee eee 188 196 
Jantiary 19201021 ees ces, paetane 195 216 
‘Januanyj26,7192 18a o no) ae eee 188 195 
Jantary 27 7192 1S iva, ak ae ee ee 187 192 
Noveniber:3, 49215...) Lane 194 193 
November 4,:1.921 5 es ee Ae ae 200 204 
Novem berzonl921ye.g corn eee 199 203 
Novembers/::192 bi, Shaner ae ene Ave 215 
Novenbern 1051921700 ee a; eae ea 202 208 
February dy 19245) fer eee. 188 194 


From these data it is seen that with complete repose the 
effort of comfortably sitting in a chair is scarcely greater 
than that of lying on a couch. From these observations as 
to the degree of repose and the position of the body, we can 
say that for basal metabolism measurements the patient 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 33 


may either le or sit in a steamer chair, well supported, so 
that there is no strain. Relaxation is the keynote. The 
aim is not “rigid” repose, but “inert” relaxation. 


PRECEDING MUSCULAR ACTIVITY 


For basal metabolism work it is also very important to 
recognize and attempt to minimize the effect of muscular 
activity preceding the measurement. The subject must 
not run or walk fast in the open air, and must not walk up 
several flights of stairs prior to the test, for muscular work 
has a pronounced after-effect. Indeed, we are beginning to 
suspect that the effort of walking on icy sidewalks or against 
a severe wind, shivering, and exposure to cold prior to met- 
abolism tests are all reflected in the subsequent metabolism 
measurements. 

FOOD INGESTION 

Muscular activity, then, is the factor which affects metab- 
olism most profoundly, but food ingestion with the resultant 
digestive activity is also an important factor. 

The fact of an increased heat production after food inges- 
tion has been known since the days of Lavoisier and Séguin, 
but it is due to the work of Zuntz and especially Rubner that 
an explanation of this increase was first offered. Rubner 
stated that the rise was caused by the specific dynamic action 
of the foodstuffs, thus designating the cause, but not really 
explaining it. Zuntz worked with large domestic animals, 
ruminants. ‘The content of the intestinal tract of these 
animals is very bulky, fibrous food which remains a long 


time in the intestinal tract and which must be worked over 
3 


34 LECTURES ON NUTRITION 


and over, pushed along the tract, and the undigested portions 
finally expelled in large fecal masses. 

Rubner found protein to have the largest specific dynamic 
action, but Zuntz, seeing that with practically protein-free 
rations there was, with ruminants, an enormous increase in 
heat output, concluded that this increase must be due to 
something other than protein. Zuntz thought of the seem- 
ingly large amount of work performed. Later, American 
| experiments showed that the use of purgatives or of agar- 
_ agar, producing a rapid succession of stools, was without 
effect on the basal metabolism of man. Dogs, whose pan- 
creas had been in large part extirpated and in consequence 
had a very low absorption for protein and fat (resulting in 
voluminous stools) showed practically no specific dynamic 
action when fed meat rations. Consequently, it was hardly 
possible to conceive of the mechanical work of peristalsis and 
evacuation as the cause of the increased heat. 

Many years ago Friedrich Miiller suggested, in a rather 
remote publication, the possibility of the increase being due 
to cell stimulus. From various reasonings it appeared that 
acids such as the amino-acid in the protein and fatty acid 
of acidosis or partly burned fat were the cause of the in- 
creased heat, inasmuch as they produced a stimulus to the 
body. Thus with the acidosis of diabetes, for example, there 
is increased metabolism. When levulose is fed, there is strong | 
' evidence that it is converted into fatty acids. This explains, 
at least in large part, the pronounced stimulating effect of 
levulose ingestion. 


With cane sugar, consisting of half levulose and half dex- 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 35 


trose, the increment is somewhat less. Finally, the large 
increase found with ruminants may be the result of the ex- 
tensive fermentations in the intestinal tract. Sixty years 
ago Herbert Grouven® in Salzmiinde proposed the theory 
that with ruminants all carbohydrates were absorbed not 
directly into the blood stream, but through fermentation 
and through the path of fatty acids. With ruminants an 
increase in the heat production with a non-protein ration 
is a fact. Mechanical movements can hardly account for it, 
and the theory of acid stimulation is probably the best ex- 
planation, since the formation of fatty acids by the fermen- 
tation of carbohydrates would result in their being absorbed 
and being carried to the cells, and there acting as a stimulus. 


PERMISSIBLE BREAKFAST PRIOR TO METABOLISM TESTS 


Under all these circumstances it is seen that the taking 
of any food (particularly protein and ketose sugars) prior 
to basal metabolism tests should be proscribed. This is the 
common practice at present, but there are two rather serious 
objections to it. In the first place, many people believe that 
going without food, even for one meal, is harmful and weak- 
ening. Hence there is often a real or fancied feeling of faint- 
ness and dizziness, which increases the apprehension felt by 
the novice in the first metabolism test. In the second place, 
the complete withdrawal of food may, with children and 
also with the obese, soon bring about an acidosis which, as 
we have seen, stimulates metabolism, so that the induced 
acidosis might increase metabolism more than the effect 
of the food. 


36 LECTURES ON NUTRITION 


For these reasons there is a certain modern tendency to 
consider the advisability of allowing a small, non-stimulat- 
ing breakfast. Du Bois® permitted a small breakfast to secure 
a feeling of satiety and repose in some very active boy 
scouts, whose basal metabolism he wished to measure, un- 
complicated by restlessness. 

At the Nutrition Laboratory, capitalizing later knowledge 
regarding the effect of special foods, Mrs. Benedict and I 
proposed a breakfast? (Table 3) characterized by the absence 
of protein and the ketose sugars and by the presence of 
fat, which gives a greater feeling of satiety. Such a meal 


TABLE :3 
NUTRITION LABORATORY PERMISSIBLE BREAKFAST 


1 cup (200 c.c.) of caffeine-free coffee. 
16 milligrams of saccharin. 

30 grams of medium cream. 

25 grams of potato chips. 


Total calorific value about 250 calories. 


has no appreciable effect on metabolism, “stays by” the 
person, and gives a feeling of euphoria that is not purchased 
at the expense of a metabolism stimulated by protein or 
ketose sugars. All technicians find that the most difficult 
day for the new patient is the first day of the tests. It would 
seem as 1f the apprehension of the first day could be lessened 
if this light breakfast were allowed. The test could then be 
repeated on the next day, with the subject in the post-absorp- 
tive condition, that is, at least twelve hours after the last 
meal, which should not have contained a large amount of 
protein. 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 37 


SPECIAL FACTORS 
There are several factors which, although supposed to have 
an effect on basal metabolism when brought to play inten- 
sively on the human organism, may not in moderate degree 
prove to be sufficiently effective to demand special attention. 


ENVIRONMENTAL TEMPERATURE 


The effect of a cold environment is the first of these factors. 
If a person is exposed to cold so that he shivers, the met- 
abolism must be greatly raised, for shivering is a muscular 
act, and as we have already seen, muscular work has a pro- 
found effect. Exposure to cold results in loss of body heat 
and a disturbance in the storage of heat. Soon the shiver- 
ing starts up and the heat production now attempts to keep 
pace with the heat loss. But in a sense this is an indirect 
effect of the cold on heat production through the muscular 
work of shivering. Does the cold per se, independent of 
shivering, affect basal metabolism? This question is still 
much discussed. The Nutrition Laboratory’s data, which 
are rather extensive, point to a certain production of heat 
without shivering, as incident to prolonged exposure to cold. 
Rubner has long maintained that there is a specific heat 
production as a result of exposure to cold. On the other hand, 
Johansson in Stockholm and Loewy in Berlin have claimed 
that there is no excess heat production without shivering. 
Johansson’s experimental data are flawless, and no experi- 
menter has ever secured a greater degree of muscular repose 
than he. Under these conditions he finds no effect of cold, 


even on the nude body. 


38 LECTURES ON NUTRITION 


If the body, as a whole, is exposed to cold air, heat is lost 
to the air. Thus, the face, hands, and neck lose heat. But 
what is the effect on the heat production? There are two 
possible means of securing temperature equilibrium: (1) By 
decrease in the blood supply to the exposed parts, until 
the temperature of the skin becomes lowered so that the 
normal heat loss is again attained, and (2) by increased 
heat production to compensate for the increase in heat 
loss. 

In the latter case the skin temperature tends to remain 
constant, and in the former it becomes lower. 

To test these points experiments have been in progress 
at the Nutrition Laboratory for several years. A subject, 
capable of withstanding prolonged exposure to cold, of un- 
usually placid temperament, and with extraordinary powers 
of relaxation and repose, was found in a very co-operative 
professional artist’s model. When the subject disrobes, little 
or no change in the basal metabolism is found at first, thus 
confirming Johansson’s short experiments. But as time 
goes on and the body and peripheral tissues become colder, 
there is a noticeable increase in metabolism long before 
shivering sets in. 

The first and almost instantaneous effect of exposing the 
previously warm, clothed body to cold is a great wave of heat 
loss, due to the great temperature potential between the skin 
(approximately 33° C.) and the environmental air (approxi- 
mately 15° C.). This pronounced heat loss is followed by a 
lowering of the skin temperature and, indeed, tissue tem- 
perature, thus reducing the temperature potential. Finally, 


BASAL METABOLISM—-MEASUREMENT, SIGNIFICANCE 39 


the skin temperature ceases to fall, and the heat loss adjusts 
itself to the prevailing temperature difference. 

A calorimeter (a so-called “emission calorimeter’’) suffic- 
iently sensitive to measure the actual heat loss in very short 
periods, of one minute or less, has only recently been available. 

The subject was placed inside of the emission calorimeter, 
covered with several layers of blanket, but otherwise nude. 
The heat loss, which was compensated by an electric current 


of measurable intensity, was established under these conditions 


CRORES 
MINUTE LOSS OF HEAT ON DISROBING 


1.8 PRS abe [kere ee 


Fig. 4.—Comparison of loss of heat before and after disrobing. From 
10:20 to 11:00 a.m. the subject was nude, but covered with blankets. At 
11:00 a.m. the blankets were suddenly removed. 


for approximately an hour or more. Then at a given instant, 
by means of previously arranged cords, the blankets were 
suddenly rolled back, thus producing the exposure of the nude 
body to the cold air. The heat lost as a result of this ex- 
posure is indicated by the pronounced rise in the curve. 
There is then a subsequent decrease in heat loss as the sur- 
face of the body cools off, until finally an approximate level 
is reached where the loss is fairly constant, but at a dis- 


40 LECTURES ON NUTRITION 


tinctly higher level than when the subject was lying, covered 
with a blanket. 

A man lying uncovered on a hot, tropical night has a rectal 
temperature probably not much different from that of the 
arctic Eskimo in his ice-lined igloo, wrapped in furs. But 
in the case of the Eskimo certainly the exposed parts of the 
body, such as the face and hands (if not protected), must have 
a much lower temperature. Is the metabolism of the man 
in the tropics different from that of the man in the arctic or 
temperate zones? This question is still being stoutly debated. 

Without considering these extremes of temperature, the 
question may properly be raised as to whether the environ- 
mental temperature should not be essentially the same in 
all metabolism measurements that are to be compared, that 
is, under conditions supposedly reproducible for basal metab- 
olism measurements? No specific requirement for environ- 
mental temperature is included in the modern stipulations 
for basal conditions. It is the common custom, however, to 
make tests at “room temperature,” presumably somewhere 
between 15° and 25° C. and it is tacitly understood by all 
workers that the subjects should be assured a comfortable 
temperature, neither too hot nor cold enough to induce shiv- 
ering. Undoubtedly too little attention has been paid to 
this point heretofore, but many of the fundamental measure- 
ments of the metabolism of human beings have been made 
with respiration calorimeters which are so constructed me- 
chanically that the environmental temperature is automat- 
ically held approximately at 20° C. 

Little, if any, attention has been paid to publishing the 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 41 


data regarding the kind and the amount of clothing and bed 
covering. For this neglect Lefévre™ has rightly criticized us. 
Indeed, Lefévre goes so far as to maintain that, owing to 
the conditions for heat loss present in practically all basal 
metabolism measurements as at present made, the heat loss 
is so great that so-called ‘‘basal’’ metabolism, as measured, 
is always higher than the true basal. Measurement of the 
true basal is secured only when every precaution has been 
taken to prevent loss to the environment. ‘This is possible, 
he maintains, only when the body is immersed in an indiffer- 
ent water-bath at 35° to 36° C., for under all other conditions 
there is an excess heat production to combat the loss to the 
cold environment. 

If Lefévre’s contention is true, then obviously current 
basal values are all too high. ‘This concept constitutes a 
serious challenge to modern metabolism measurements. 
Although at the Nutrition Laboratory we were convinced 
that Lefévre had fallen into the common error of so many 
critics of not clearly differentiating between heat loss and 
heat production, the criticism of modern metabolism technic 
seemed to us too plausible to go unchallenged. Accordingly, 
Mrs. Benedict and I have made a series of tests* with sev- 
eral subjects, in which the basal metabolism was first meas- 
ured while the subjects were lying, clothed and lightly cov- 
ered, in the laboratory, in a rather unusually low room tem- 
perature of 15° C., to accentuate, if possible, the influence 
of the cool environmental temperature. The. subjects were 
clothed as ordinarily and covered with one thickness of a 
light cotton blanket. Immediately after the measurements 


42 LECTURES ON NUTRITION 


under these conditions the subjects entered a neutral bath 
(35° C.) in a very warm room (30° C.), and several metab- 
olism experiments were made under these conditions. 

Since all the subjects were well trained to metabolism 


measurements, the basal values found prior to the bath show 


Fig. 5.—Measurement of the oxygen consumption of a subject during 
immersion in a water-bath. 

reasonably close agreement with each other. After the sub- 

jects entered the bath the metabolism measurements, in- 

stead of decreasing, as predicted by Lefévre, almost invari- 

ably tended to increase slightly. The results are shown 

in Tables 4 and 5. 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 43 


TABLE 4 


Mr. B., AGE ForTY-SEVEN YEARS; NUDE WEIGHT, 67 Ka.; HEIGHT, 
169 CM. 


(Oxygen consumption in cubic centimeters per minute) 


Date. HES ae ae aoe Bath at about 36° C. 
October 4th 204 ae 205s 197 208 215 210 202 
October 5th 203 207 200 21152222235 224 230) 
October 6th 19682 2209 1213 Zea oPalo 921236) 224 
TABLE 5 
Miss W., AGE THIRTY-THREE YEARS; NUDE WEIGHT, 60 Kc.; HEIGHT, 
ar nibh 162 Cm. 


Oxygen consumed per minute. 


Room | ——— | Tempera- 
Date. tempera- Basal ture of 

Sr (average), Bath, c.c. eda 

Cc, 

December 7th 2150 192 219 «210 186 SHild) 
December 8th 15.5 190 194 186 191 190 36.3 
December 10th | 16.2 194 182 200 206 187 36.5 
January 3d Se 192 195 195 197 $9.0 
January 24th 14.8 198 194 200 192 196 34.0 
January 25th 14.8 198 208 201 Oo 
January 28th 15.1 200 191 193 206 38.0 


From these tests one can conclude that an environmental 
temperature of 15° C. or above, when the subject is clothed 
and lightly covered, does not raise the metabolism, or at 
least the metabolism is not lowered by subsequent complete 
immersion in a neutral bath of 35° to 36° C. Consequently 
a room temperature of 15° C. (if the subject does not feel 
cold and is protected from drafts) is a suitable thermal con- 


A4 LECTURES ON NUTRITION 


dition for making basal metabolism measurements. Im- 
mersion in a water-bath at 35° C. is neither practical nor 


effective in lowering the metabolic rate. 


FEVER 


The important findings of Du Bois’ on the influence of 
body temperature elevation on metabolism should not be 
disregarded. Measurement of the body temperature, pref- 
erably rectal, should assure the operator that a body tem- 
perature prevails within normal limits, taking into consid- 
eration the well-known diurnal temperature variations. 
Slight elevations in body temperature may make a metabolism 
measurement worthless for comparative purposes. 


MENTAL ATTITUDE AND PYSCHIC REPOSE 


While the two most pronounced factors affecting metab- 
olism are food ingestion _and.muscular work, the factor of 
temperament or mental repose may be of significance, al- 
though it may be secondary, acting through the muscles. 
If the subject is nerved up, apprehensive, and irritable, this 
condition contributes to an increased metabolism. For 
example, one of our subjects came to the laboratory once 
seemingly very sleepy and exhausted. To our surprise his 
metabolism was high, but it was found that a conflict the 
night before with an irate father, with whose daughter he 
had tried to elope, had left a somewhat shattered nervous 
system with an increased metabolism that showed above the 
seeming muscular exhaustion and somnolence. On two in- 
stances also we have had to contend with the after-effects 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 45 


of an alcoholic night with some of our subjects. Such orgies 
raised the metabolism. 

Unquestionably apprehension and fear raise metabolism. 
Hence all methods to depress such apprehension are justi- 
fiable. As already pointed out, the steamer chair as a sub- 
stitute for the bed and the permissible breakfast may both 
produce a feeling of euphoria, allay fear, and banish’ appre- 
hension to such a degree as to warrant their intelligent use. 
The long wait prior to metabolism tests, the sight of seem- 
ingly complicated apparatus, the attachment of nosepieces, 
mouthpiece, or face mask, should all occur under the least 
irritating conditions and in the shortest time possible. 


SLEEP 


Under conditions of sleep one would expect to have the 
greatest degree of muscular and psychic repose. Hence the 
influence of sleep on metabolism possesses unusual interest 
for us. This problem is not a simple one to study, for with 
the onset of sleep codperation on the part of the patient 
disappears, but also (it should be equally emphasized) an- 
tagonism disappears. Consequently experiments on the 
effect of sleep must, for the most part, be made in some form 
of respiration chamber where mouthpiece, nosepieces, or 
mask are not employed. With an especially well-trained 
subject it. is not impossible to make such observations. The 
Nutrition Laboratory has certain data with regard to sleep, 
as yet incomplete, which justify us in stating that the in- 
fluence of sleep is very pronounced on the mechanics of res- 
piration, causing transitory and perhaps permanent changes 


46 LECTURES ON NUTRITION 


in ventilation rate and the storage of carbon dioxide in the 
body. 

Whether sleep affects the total metabolism is by no means 
clearly established, although during sleep there is a distinct 
tendency towards a lower metabolic level. Hence for prac- 
tical purposes and for reproducible conditions it is clear that 


SUBJECT D, APR. 14, 1916 
500 c.c. NaCl SOLUTION (0.6p. ct) MASK 


HALF HOURS AFTER RECTAL INJECTION BEGAN 


Fig. 6.—Respiratory quotient, oxygen absorption, and pulse rate of 
Subject D, April 14, 1916, before and after rectal injection of 500 c.c. 
0.6 per cent. solution of sodium chloride. 


metabolism measurements should only be made with the sub- 
ject awake. 

The curve in Figure 6 (one of many taken from a forth- 
coming monograph by my associate, Dr. T. M. Carpenter) 
shows two striking effects of sleepiness: first, the fall in 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 47 


the pulse rate, and second, the profound disturbance in 
the respiratory quotient. The absence of sleep is insisted 
on to such an extent in the Nutrition Laboratory that Dr. 
Carpenter frequently arranges a signal magnet near the ear 
of the subject, which gives a slight sound at the end of each 
half minute, and the subject is provided with a push button 
to register his reaction to this. The absence of registration 
is an indication either of drowsiness or of complete sleep. 
While it is true that most patients will be too alert psy- 
chically to sleep, not infrequently drowsiness does occur. 
In all such cases we believe it is necessary for the operators 
to insure that their patient is awake during respiration ex- 
periments. This is particularly the case if the respiratory 


quotient is to be studied. 


CONDITIONS SPECIFIED FOR METABOLISM MEASUREMENTS 


With a consideration of the foregoing factors, which are 
known or supposed to influence basal metabolism, we are in 
a position to specify perhaps more exactly than before the 
best and most practical reproducible conditions for measur- 
ing basal metabolism. 

The first and foremost requisite is repose. The subject 
should be in the greatest degree of relaxation, either lying 
on a couch or in a steamer chair. The injunction for com- 
plete muscular repose should not be so strict, however, as 
to result in the person being cramped or muscle-bound. 
A graphic tracing of the activity, with the employment of a 
pneumograph either about the thighs or under the bed spring, 
is helpful. 


48 LECTURES ON NUTRITION 


The second requirement is the post-absorptive condition. 
No food should have been eaten for twelve hours and the 
last meal should not have been excessively high in protein. 
It is doubtful if a special diet for several days prior to the test 
is of real value. With the demonstrated absence of the stim- 
ulating effect of a permissible breakfast, such a light meal 
can be given, although at present it will be best to give it 
only on the first day of the test. If the second day’s meas- 
urements are lower than the first, these values should be used, 
for in general it should be stated that, barring technical errors, 
the lowest values found represent basal metabolism and all 
other values are too high. 

The subject should be comfortably clothed and not too 
warm or too cold. The amount of protection should be such 
as to secure the greatest degree of relaxation. 

Every effort should be made to combat apprehension by 
avoiding an undesirably long wait prior to the tests and 
unnecessarily prolonged arrangement of apparatus, mask, 
mouthpiece, or nosepieces. A quiet, placid relaxation is ideal. 

The body temperature (if feasible, the rectal temperature 
should be measured; if not, the buccal) should be within 
normal limits. If morning buccal temperatures are over 
99° F., the time should not be wasted on a basal metabolism 
measurement. 

The subject should be kept awake. A signal with a push 
button response is desirable, not only as a proof that the 
subject is awake, but because it is a good thing psychologic- 
ally to have the patient’s mind on some simple, non-muscular | 
task. 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 49 


STANDARDS OF BASAL METABOLISM 


If the metabolism is measured under such stipulations, 
one can consider it either basal or at least standard, for the 
conditions are standardized and are reproducible. Measure- 
ments made under similar conditions are, moreover, logically 
comparable. Whether the measurements are truly basal or 
not may still be debated. Certainly with complete muscular 
and mental repose, without food in the stomach for at least 
twelve hours prior to the measurement, with a comfortable 
environmental temperature, without a febrile temperature, 
the only factors capable of lowering the metabolism under 
these conditions are deep sleep and prolonged fasting or 
undernutrition. Bearing in mind that the chief object in 
basal metabolism measurements is to secure reproducible, 
comparable conditions, it would seem as if the above speci- 
fications are all that should be insisted on at the present 


time. 


SEASONAL VARIABILITY 


Are the results obtained under these conditions comparable 
and are they comparable with the rather considerable number 
of basal metabolism measurements heretofore published? 
In other words, is the standard metabolism as measured 
constant? This question applies, first, to the individual. 
Since under the specified conditions of measurement the 
immediate factors influencing basal metabolism have been 
ruled out, the doubtful element is primarily only the matter 
of season. Measurements made at night on a group of 


young men inside of a respiration chamber at the Nutrition 
4 


50 LECTURES ON NUTRITION 


Laboratory did show a tendency for a decrease in metabolism 
between the first of October and the first of January.’ It 
is also possible that the same individual will have a very 
different metabolism in the arctic or temperate zones than 
he will after a sojourn of several months in the tropics, but 
this is not as yet definitely established. 

For all practical purposes one can conclude that with the 
same individual the basal metabolism remains reasonably 
constant from day to day and even from season to season. 
That there are not profound influences due to considerable 
changes in habits of life is not as yet disproved. The labora- 
tory dog of Lusk, after a sojourn in the country, showed an 
increased metabolism of 20 per cent, although there was no 
change in weight. This fact suggests that the effect of re- 
cuperation after a summer’s holiday is well worthy of further 


investigation. 


COMPARISON OF THE METABOLISM BETWEEN INDIVIDUALS 


It is of greatest importance to the clinician, however, to 
know whether the basal metabolism as measured on one indi- 
vidual may be compared intelligently with that measured 
on other individuals or groups of individuals. Is it not pos- 
sible to find some method of intelligently comparing the meas- 
ured metabolism of a patient with the general trend of metab- 
olism of other individuals of similar age, height, weight, and 
sex? ‘This has been attempted in several ways. One of the 
earliest methods was to compare the metabolism on the basis 
of the body weight, on the assumption that a large person 
would have a larger heat production than a smaller person. 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 51 


Hence it was argued that the more rational method of com- 
parison was on the basis of the heat production per kilogram 
of body weight. This comparison, however, is, at least 
with adults, fundamentally wrong, inasmuch as it assumes 
that each kilogram of body substance has exactly the same 
heat-producing power, whereas we know that inert fat is 
not comparable to active muscle as a producer of heat. 

Another comparison which has had a most pronounced 
influence on the interpretation of metabolism measurements 
was that early recognized as the relationship between the 
heat lost from the surface of the body and the surface area. 
It was found, as would be expected, that the heat loss (about 
1,000 calories for each square meter every twenty-four hours) 
was approximately proportional to the surface area, not only 
with different individuals of the same species and different 
size, but likewise between different species. The ‘‘surface 
area law,” therefore, became one of the most important con- 
tributions to our knowledge of the physiology of energy 
transformations. 

From the practical standpoint it is perhaps not a matter 
of importance whether the rate of metabolism is directly 
proportional to the surface area, whether it is controlled by 
the heat loss from the body, whether the heat loss is inde- 
pendent of the heat supply, or whether the heat produced is 
determined by the active mass of protoplasmic tissue and 
the stimulus to the cells. But it is important to know whether 
there is a referable basis which can be used intelligently for 
comparing various individuals. 

At the outset it may be stated that the original surface 


52 LECTURES ON NUTRITION 


area law, as outlined by Rubner, implied that the metabolism 
was proportional to the surface area, and that this law was 
subsequently extended throughout almost the entire animal 
kingdom and has since been erroneously extended to cold- 
blooded animals. Its application to human beings has al- 
ready been limited by common consent in two ways, in that 
an equal surface area is considered to have a different value 
between men and women and a different value between the 


old and the young. 
The Nutrition Thee s data for children and adults 


are shown in Figure 7 on the basis of the heat production 
for each square meter of body surface every twenty-four 
hours. Here the influence of sex is shown throughout prac- 
tically the entire life, except in the early months, and the 
influence of youth is evidenced by the extraordinarily low 
metabolism at birth or shortly after, with a maximal metab- 
olism at about the age of one year. 


CURRENT STANDARDS OF REFERENCE 


There are in current use two standards of reference.* 
One is based directly on the surface area, making allowance 
for sex and age. This standard of reference is extremely 
simple and very practical, and the medical profession and 
physiologists as a whole owe a great debt to Dr. Eugene F. 
Du Bois! for it. The rule-of-thumb procedure of the earlier 
surface area estimations is avoided in this standard by in- 


cluding the remarkably accurate measures of the surface 


* The standards of Dreyer (Lancet, 1920, Part 2, p. 290) are not here | 
considered, although attention should be called to this work with its pos- 
sible potentialities. 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 53 


area of the human body introduced by Du Bois and Du Bois.’ 
With such measured surface areas, or with surface areas 


BOYS AND MEN —-~—-~-——-—= 


= 
i 
= 
2) 
= 
(a) 
Zz 
< 
co) 
3 
as 
Oo 


Fig. 7.—Comparison of the basal heat production of children and adults per square meter 
of body surface per twenty-four hours referred to weight 


derived from their simple formula, one can, by knowing the 
surface area, age, and sex of the individual, compare the 


54 LECTURES ON NUTRITION 


measured metabolism with a standard which is now ex- 
tensively used, the so-called Du Bois standard. 

The Nutrition Laboratory, believing, as a result of more 
intimate biometric analysis, that the factors affecting metab- 
olism independently are sex, age, weight, and height, and 
dealing for the most part with physiologic rather than path- 
ologic problems, has contended that a biometric formula 
involving these four factors is scientifically better founded. 
Hence we have the prediction formulas printed in collabora- 
tion with Professor J. Arthur Harris! of the University of 
Minnesota: 


Formens iy dy tant h = +66.4730 + 13.7516w + 5.0033s — 6.7550a 
For women......... h = +655.0955 + 9.5634w + 1.8496s — 4.6756a 
h = total heat production each twenty-four hours. 

w = weight in kilograms. 
s = stature in centimeters. 
a = age in years. 


Innumerable comparisons of these two methods of stan- 
dardization have been made, but when one considers that 
both standards were based in large part on the same experi- 
mental material, that is, on the Nutrition Laboratory meas- 
urements, it is not surprising that they agree so well. 

Any logical basis for the use of the surface area law in 
medicine is enormously complicated by the fact that the 
adherents of the surface area law stoutly maintain that it 
was never meant to apply to other than definite physiologic 
conditions. Yet they do not hesitate an instant to refer 
measurements in pathologic cases (in which obviously under- 
nutrition and at times fasting necessarily occur) to normal 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 55 


standards, and because they find the metabolism for each 
square meter of surface area is the same as the standard, 
they do not hesitate to pronounce it normal, overlooking 
entirely that a lowered metabolism due to undernutrition may 
be accompanied by a superimposed effect of some disease. 

It is more than likely that the time is now at hand when 
definite quantitative attention must be paid to the state 
of nutrition of various patients, in interpreting their basal 
metabolism. The difficulty of securing any uniformity in 
the method of indicating the state of nutrition is very pro- 
nounced. The Nutrition Laboratory has been quite inclined 
to accept: Pirquet’s pelidisi as one of the best numerical 


Pelidisi of Pirquet 


AY, 10 X weight (gm.) 


sitting height (cm.) 


indices thus far available, leaving to medical men the value 
of his subsidiary observations with regard to the turgor, 
amount of body fat, and blood supply to the skin. 

In a recent analysis of the metabolism of a large number 
of girls and women it was found that when the measured 
twenty-four-hour heat production was divided by the pelidisi 
of these individuals, a straight line function was found, begin- 
ning with children as young as three months and extending 
to old age.? It would thus appear as if many of the aber- 
rant deviations from the general trend of metabolism measure- 
ments, noted particularly with girls and women, disappear 
when the state of nutrition, as indicated by the pelidisi, is 
taken into consideration. 


56 LECTURES ON NUTRITION 


It is clear that the state of nutrition of the individual 
is pronouncedly reflected in the general metabolism. During 
complete fasting the metabolism is very low. During under- 
nutrition it is likewise very low. With surfeit feeding it is 


TOTAL HEAT 
Gals PELIDISI REFERRED TO HEIGHT GIRLS 
Ee 


HL 


20 
60cms. Wi 


Fig. 8.—Relationship between SING: and the basal heat production 
each twenty-four hours divided by the pelidisi, with females from one 
week of age to full maturity. The hollow dots represent girls below 10 
kilograms in weight or younger than one year; the solid dots represent 
girls 10 kilograms or above in weight; the crosses represent groups of 
girl scouts, twelve girls in each group; and the arrow indicates the average 
value for 103 adult women. 


very high. All of these conditions are entirely distinct and 
separate from the immediate effects of food ingestion. The 


fact that so-called “hospital normals” have a low metabolism, 
the fact that Lusk’s dog, when confined to the laboratory 


BASAL METABOLISM—MEASUREMENT, SIGNIFICANCE 57 


for several months, had a low metabolism, the fact that a 
large group of students subsisting on half rations had a very 
low metabolism, coupled with the fact that by excessive 
feeding there may be an increased metabolism, such as to 
strengthen belief in Voit’s old idea of “luxus consumption” 
(so strongly advocated at the present day by Grafe), are of 
themselves sufficiently important to indicate the basic sig- 
nificance of the “‘state of nutrition.” 

Basal metabolism is reasonably constant in the same in- 
dividual. It is also reasonably comparable between different 
individuals, if weight, height, sex, and age are taken into 
consideration and provided that the pronounced influence 
of under- or overnutrition is borne in mind. It is not 
fixed or determined by surface area and the loss of heat to 
the environment. While undeniably in a state of flux and 
subject to the influence of other factors than those of age, 
size, and sex, it still remains a fact that basal metabolism 
(depending on heat production and not heat loss) is a very 
good index of the general state or level of vital activities as 
produced by the mass of the cells and the stimulus to their 
activities. 

Modern, simplified methods make possible the determina- 
tion of basal metabolism with a technic that is much easier 
than the technic for counting the blood corpuscles, and with 
an instrument that is not appreciably more expensive. In 
fact, it is not perhaps too speculative to state that the present 
wave of enthusiasm for basal metabolism measurements and 
their application primarily to endocrine disturbances may 
in time be replaced by a more rational use of this important 


58 


LECTURES ON NUTRITION 


measurement as an index of general vigor, tone, and physio- 


logic state. 


NR = 


BIBLIOGRAPHY 


. Aub and Du Bois: Arch. Intern. Med., 1917, 19, p. 831. 
. Benedict: Proc. Am. Philos. Soc., 1924, 63, p. 43. 
. Benedict and Benedict: Boston Med. and Surg. Jour., 1923, 188, 


p. 849. 


. Benedict and Benedict: Bull. Soc. Sci. d’Hygiéne Alimen., 1924, 12, 


pp. 480 and 541. 


. Benedict, Miles, Roth, and Smith: Carnegie Inst. Wash. Pub. No. 


280, 1919, p. 497. 


. Du Bois: Arch. Intern. Med., 1916, 17, p. 887. 
. Du Bois: Journ. Am. Med. Assoc., 1921, 77, p. 352. 
. Du Bois and Du Bois: Arch. Intern. Med., 1915, 15, p. 868; Sawyer, 


Stone, and Du Bois, Arch. Intern. Med., 1916, 17, p. 855; Du Bois 
and Du Bois, Arch. Intern. Med., 1916, 17, p. 863. 


. Grouven: Physiologisch-chemische Fiitterungsversuche. Zweiter 


Bericht iiber die Arbeiten der agrikulturchemischen Versuchssta- 
tion zu Salzmiinde, Berlin, 1864, p. 505. 


. Harris and Benedict: Carnegie Inst. Wash. Pub. No. 279, 1919,p. 227. 
. Harris and Benedict: Carnegie Inst. Wash. Pub. No. 279, 1919. 

. Lefévre: Bull. Soc. Sci. d’Hygiéne Alimen., 1922, 10, p. 595. 

. Lefévre: Bull. Soc. Sci. d’Hygiéne Alimen., 1922, 10, p. 601. 

. Lusk: Journ. Biol. Chem., 1915, 20, p. 565. 

. Miiller: Volkmann’s Sammlung klin. Vortrige, Leipzig, 1900, No. 272, 


p. 44, 


PROBLEMS OF METABOLISM 


GRAHAM LUSK 


In 1870, fifty-five years ago, Carl Voit® wrote an article 
of a hundred pages entitled, ‘“The development of the doc- 
trine of the source of muscular energy and of the doctrines 
of nutrition during the past twenty-five years.” He is re- 
sentful of the criticisms of Liebig, who was then sixty-seven 
years old and no longer actively experimenting. He points 
out that Liebig himself in 1844, twenty-six years before, had 
denounced Berzelius, who at that time happened also to be 
sixty-seven years old, on the ground that Berzelius had 
ceased to experiment, devoted his entire intellectual powers 
to theoretic speculations, and was therefore not competent 
to criticise the experimental work of others. Voit closes 
his paper with the following words: 

“While preparing this defense of my viewpoint to which I 
have been driven, there has grown upon me the conviction 
that the knowledge of nutrition is in a state of unhindered 
and flourishing development. The ship which for a long time 
was only moved by cross currents to and fro, because it did 
not have the rudder called ‘experiment,’ now moves for- 
ward uninterruptedly on its course. Let those who desire 
to remain uncertainly adrift continue this attitude, but they 


will be outdistanced and they will have no part in the re- 
59 


- 


60 LECTURES ON NUTRITION 


ward when the other richly laden vessel returns to the home 
port.” ) 

Such words clearly emphasize that only by the. experi- 
mental method can one hope for progress and success. Also, 
one of the fundamental necessities in all scientific work is 
to know the charted sea. I would suggest a better knowl- 
edge of the old-time literature. Last year I gave to Robert 
Weiss, a pupil of Biedl’s, an article written by Voit, but 
published in my name in the Zeitschrift fiir Biologie, vol. 27, 
1890, entitled “Ueber den Einfluss der Kohlehydrate auf den 
Eiweisszerfall.’’ Weiss returned the volume to me with the 
remark, ‘It is all absolutely modern.”’ It seems to me that 
perhaps the only advantage of advancing age is that one is 
able to recall what the old masters thought. One has but 
to go back to the work of Liebig to find ideas that one natu- 
rally attributes to Rubner, or to read the work of the early 
French school of eighty to a hundred years ago to see where 
Liebig received his inspiration. When Lafayette Mendel 
and I were young men together in New Haven I called his 
attention to Voit’s “‘Stoffwechsel und Ernahrung,”’ which he 
has since read many times and which still deserves atten- 
tion, not only from us in America but also from many in 
Germany where it is well-nigh forgotten. Modern workers 
are not sufficiently well grounded in the older literature of 
their subjects, and it seems that this is one of the “prob- 
lems of metabolism” which cries out for treatment and for 
cure. It appears to be a need in all countries. 

That the new is not always the most illuminating may be ~ 
learned by reading Billroth’s “The Medical Sciences in the 


PROBLEMS OF METABOLISM 61 


German Universities,” which, though written fifty years ago, 
has just been translated from the original German. The 
problems confronting us are the same as those of the German 
universities half a century ago. 

A well-nigh indispensable help to research is criticism; 
it provokes better work. Well-trained men who settle alone 
in a community in which there is no searching criticism are 
likely to suffer a lowering of their intellectual morale. The 
right kind of criticism brings out appropriate response. 
Spoken criticism is usually not as effective as written criti- 
cism, for the latter exerts pressure from a wide audience. 
One will not have it said of him 


““ “Tis strange the mind, that very fiery particle, 
Should let itself be snuffed out by an article.” 


Your victim cries, ““You have forced me to write another 
book’; and you may answer, “It is well.” 

The worst thing one may say about a laboratory worker is 
that his work is sloppy. It is sometimes necessary to say it. 
The literature is burdened with poor work. Sometimes 
silence is sufficient. 

It should be no affront to say that theories are false, and 
yet a scientist is often most sensitive in the region of his 
theories, and the wound may be deep. One is reminded 
of the blind men of Hindustan who were taken to visit an 
_ elephant. One of them handled the trunk and found the 
elephant to be very like a rope, another felt the leg and 
found the elephant to resemble a tree, and so on. 


62 LECTURES ON NUTRITION 


“So these wise men of Hindustan disputed loud and long, 
Each to his own opinion exceeding stiff and strong, 
Though each was partly in the right, 

Yet all were in the wrong.” 


I remember poking fun at the tomato as being nothing 
but water colored red, only to be later shown that it con- 
tained vitamins A, B, and C. All publications should con- 
tain paragraphs devoted to “the errors of the author and 
his critics.’ Criticism which is personally unkind, as was 
that which flourished a hundred years ago, has no place in 
the world today. Such criticism perhaps reached its climax 
in Byron’s picture of the fall of Southey into Lake Avernus, 


“He first sank to the bottom like himself, 
And then rose to the top, like his works, 
For all corrupted things are buoyed up like corks, 
By their own rottenness.”’ 


We have today no Vision of Judgment which delineates 
the ultimate repute of our own endeavors. 

In his introductory lecture to his students Carl Voit al- 
ways told them, “I do not ask you to believe anything I 
tell you because I say it is so. I only ask you to believe 
those things which I can prove to you are true.’”’ And so 
in this lecture I will try to distinguish between known facts 
and the things of which dreams are made. 


METABOLISM 


The chemical and energy transformations in living things 
constitute the basic factors in metabolism work. The scope 


PROBLEMS OF METABOLISM 63 


is wide as life itself. The anatomy of the body is well known; 
the chemical anatomy of its constituent parts is becoming 
slowly known. Witness the epinephrin of Abel and Taka- 
mine, the thyroxin of Kendall. We rejoice in Levene’s dis- 
covery of the chemical structure of nucleic acid. But when 
we contemplate the fact that blue eyes are not inherited ex- 
cept from blue-eyed ancestors, we are confronted with a 
problem transcending the scope of our analysis. The mere 
mention of such a problem indicates that the field to be tilled 
is limitless. 

About a dozen years ago the expression basal metabolism was 
introduced into the literature as representing a translation 
of the German word Grundumsaiz employed by Magnus- 
Levy. It represents the heat production of a quiet individual 
eighteen hours after food ingestion and in an environment 
which is free from thermal stimulation. Cold must not play 
on the skin, nor external heat raise the temperature of the 
body. Under these conditions the heat production mani- 
fests a marvelous constancy. During a period of eleven years 
the basal metabolism of E. F. Du Bois varied from the aver- 
age by a maximum of +8 per cent and showed an average 
variation of only +3.4 per cent. The basal metabolism of 
a dog confined in a cage and maintained on a daily diet 
sufficient to supply its needs is even more constant than this. 
Thus Rapport? in our laboratory found in Dog 19 an aver- 
age basal metabolism of 16.52 calories an hour in fourteen 
determinations during a period of fifteen months. The maxi- 
mal variation was +3 per cent and the average variation 
was less than 1 per cent throughout the whole period. Since 


64 LECTURES ON NUTRITION 


eight alcohol checks, made in order to measure the accuracy 
of the respiration calorimeter during this period, showed a 
maximal error of +1.9 per cent, it is evident that the slight 
variations in the determination of the basal metabolism of 
the dog may in part be due to the limitations in the accuracy 
of the method employed. 

It is very important to realize this background of funda- 
mental biologic behavior. A little while ago, at one of our 
scientific meetings, a report was made of enormous day- 
to-day fluctuations in the basal metabolism of dogs. When I 
protested against this work the representative of a well-known 
laboratory rose to concur with the person who had obtained 
the hugh gyrations shown in the level of the basal metab- 
olism of the dogs under consideration. I could only hold 
my peace and wait. 

It is of utmost importance to realize the constancy of the 
background. Resting quietly in a box at the warm tempera- 
ture of 26° C. (77° F.), a dog produces from chemical energy: 
(1) electric currents at each contraction of the heart or of 
the muscles of respiration; (2) mechanical energy when the 
heart places blood under pressure in the arteries and when: 
the respiratory muscles act as power on levers, the ribs, and 
(3) the power which maintains the vibratory movements in 
the various organs of the body, the sum total of which we 
call life. The basal metabolism measures in terms of heat 
production, that is, in calories, the sum of all these various 
physical movements of animate matter. The movements 
which are supported at the expense of the oxidation of fuels 
in the body are themselves converted into heat. That the 


PROBLEMS OF METABOLISM 65 


amount of energy necessary to maintain life is a constant is 
a fundamental fact of great biologic importance. I pur- 
posely reiterate this. 

Against this constant background may be contrasted the 
results of food ingestion, of temperature influences, and of 
mechanical work, factors recognized since Lavoisier. 


VARIABILITY OF HEAT PRODUCTION 


Rubner’s experiments on the influence of environmental 
temperature on the heat production of a dog are classical. 
In the presence of cold the heat production increases to meet 
the heat lost from the body. One cannot observe a cat fast 
asleep in the sun on an icy winter day without realizing 
the important effect which cold on the surface of the skin 
must have on the heat production of the animal. 

Rubner®? has recently made important contributions to 
this subject, which deserve to be well known. In the first 
place he shows that when various forms of life are exposed to 
a temperature of 16° C., the heat production for each kilo- 
gram of body substance is extremely variable. 


METABOLISM AT UsuAL Room TEMPERATURE 
Each kilogram at 16° C. 


Weight. Calories in twenty- 
four hours. 
sina fistten ity ie auras vio at dak 1.75 gm. 39 
FY. CST ACe LIS Ne ey scaler chirrate arene nails lage 73 
WIGUSEC a4). Gear en Pe ae Se 1.75 gm. 977 
CGHINEa Dig elie. oot tae ers orene i ats 50 gm. 286 
PLE OLBE Ts Vie: erste otal, 5 vote & Sos 450 kg. 15 


A mouse living in air at a temperature of 16° C. produces 


twenty-five times more heat than a fish of equal weight. 
5 


66 LECTURES ON NUTRITION 


Rubner investigates the application of the surface area law in 
fish, amphibians, and reptiles, and finds that it does not hold. 


BASAL METABOLISM OF COLD-BLOODED ANIMALS DETERMINED AT 16° C. 


Calories for 


Calories for 
each square 


each square 


Fish, gm meter of Weight, gm meter of 
aca surface in Sgt surface in 
twenty-four twenty-four 
hours. hours. 

0.5 38.8 Frogs and toads 128 
ZiZo 44 .3 Lizard (lacerta) 45 
3.8 26.0 Alligator 47 
193 .0 30.4 Lizard (uromastix) 29 
245 .0 G225 Frogs 98 
Turtle 64 

Average 33 .08 Average 68.5 


The muscular mechanism varies in different cold-blooded 
animals. A salmon is very active in water just above the 
freezing-point, whereas snakes and frogs are listless at this 


temperature. 


RELATION OF HEAT PRODUCTION TO SURFACE AREA 


The picture of the relation of the heat production to the 
surface area changes completely when one considers the 
relations existing between various mammals. Rubner’s fig- 
ures are as follows: 

Calories for each 


square meter of 
surface in twenty- 


four hours. 
Maree Boke eat late ee lee belts aie met eae 1042 
PIS INS fo clelols ee aie. lels Gan GPS ei the ee nett ATS 1078 
Dag heres eo casts oe an eae 1039 
BRAG DIC Me ae eo ecg ne Gy Pee nee eee 917 
Guin€a pig ve cea iene a eeale ames 1131 


Mouse gic cigs aie eo cin ee Nae a eee) Tae aie 1181 


PROBLEMS OF METABOLISM 67 


These comparative figures are valid, Rubner remarks, 
despite the criticism which some individuals feel themselves 
forced to make. Why is it that the development of a species 
is so ordered that its requirement of energy is mathematically 
proportional to the surface area? If surface area is not re- 
lated to the quantity of energy produced, what is it that 
brings about these definitely ordered relationships? 

Considering the development of offspring, Rubner points 
out that the aim of growth is to produce a new organism akin 
to the parent, not only in form and size, but also similar in 
energy production, in consequence of which a gradual reduc- 
tion in the heat produced for each kilogram must accom- 
pany growth. When the child reaches the same size and 
shape as the parent, it will also have the same energy pro- 
duction for each square meter of surface, even though the 
surface area has no constant influence on metabolism. The 
surface area may be a valid method of measuring metab- 
olism even though it may not be the direct cause of the 
heat production. Also the cell mass of animals of different 
sizes manifests an adaptation to different surface areas. 
The process of manufacturing a warm-blooded animal from 
a cold-blooded one is thus pictured by Rubner: 

1. There would be a development of feathers or hair and, 
in mammals living in cold water, of subcutaneous fat, all of 
which are bad conductors of heat. Further, the physical 
regulation of body temperature would be established, by 
which the distribution of blood at the surface of the skin is 
regulated in order to control the loss of heat. The surface 
area here becomes a factor in the behavior of the animal. 


68 LECTURES ON NUTRITION 


In hot weather increased evaporation of water from the 
lungs and skin would cool the body, a mechanism possessed 
even by frogs and turtles. Through the evaporation of water 
a frog exposed to a temperature of 30° C. maintains a body 
temperature of 20° C. An animal provided with the fore- 
going mechanical arrangements may maintain the normal 
body temperature of a warm-blooded animal if the sur- 
rounding environment has a sufficiently high temperature. 

2. To complete a warm-blooded animal, however, requires 
the creation of a neuroregulatory center in the brain in control 
of the mechanism of chemical regulation by which heat pro- 
duction is increased in the presence of cold. Without this 
mechanism a warm-blooded animal would be a very incom- 
plete creature whose existence would be strictly limited to 
life at a high environmental temperature, and when exposed 
to cold would not be able to maintain its heat production 
except through muscular movements. 

We recall the cat basking in sunshine in zero weather and 
contrast its life with that of the alligator, which would freeze 
under like conditions. Rubner compares the metabolism of 
a marmot weighing 3.15 kg. during its winter sleep when its 
body temperature was 10° C., with that of the same animal 
awake with a body temperature of 36.7° C. 


METABOLISM OF A HEDGEHOG 


Body tem- Calories for Calories for each square 

perature. each kilogram. meter of surface. 
Asleep....... 10° C. vA best 47.5 
Awake...... SOc Cs 67.74 1160.0 


At a body temperature of 10° C. the marmot had a heat 
production for each square meter of surface comparable to 


PROBLEMS OF METABOLISM 69 


that of an amphibian or a reptile, but when its body was 
warmed to the normal mammalian temperature the heat pro- 
duction rose twenty-fivefold and conformed to the usual surface 
area standards, as given by Rubner. The figures recall the 
comparison between the fish and the mouse of equal weight. 

I have spent some time on this subject because the sur- 
face area standard as a measure of the basal metabolism has 
been met with flat denial in certain distinguished quarters 
without, it seems to me, giving due weight to all the facts 
of the case. I would have you bear always in mind the 
constancy of the basal metabolism and its general conformity 
to the law of surface area. 


EFFECT OF PROTEINS AND PROTEIN DERIVATIVES ON HEAT 
PRODUCTION 

The ingestion of protein always, and of fat and carbo- 

hydrate when given in quantity, increases the heat produc- 

tion. For example, I would call your attention to the follow- 

ing results taken from the work of Weiss and Rapport’ when 

Dog 19 received the same diet factors on different occasions: 


Increase over basal of 


Date. Calories. 16.48 calories, per cent. Food, gm. 
March 21, 1923 19.98 2152 Glycine, 10 
December 10, 1923 19.99 21.4 Glycine, 10 
March 7, 1924 20.05 ZU} Glycine, 10 
February 9, 1923 21.19 28 .6 Gelatin (6 gm. N) 
April 30, 1923 21.41 29.9 Gelatin (6 gm. N) 
December 7, 1923 21.62 31.2 Gelatin (6 gm. N) 
March 18, 1924 21.01 2235 Gelatin (6 gm. N) 


21.31 29.3 


70 LECTURES ON NUTRITION 


This dog, with a body weight of 9 kg., when given 10 gm. 
of glycine or 0.1 per cent of its weight, reacted on three 
different occasions, two of which were a year apart, so that 
the heat production rose by 3.50, 3.51, and 3.57 calories. 
The average is 3.527 calories, with a maximal variation of 
slightly over 1 per cent. After giving 38.7 gm. of gelatin to 
the same dog on four different occasions the average increase 
in the heat production was 4.80 calories, with a variation 
of +6.6 per cent. These experiments were made at dif- 
ferent intervals during a period of thirteen months. 

In two other experiments 43.7 gm. of casein reacted to 
increase the dog’s heat production by 5.06 calories within a 
+ variation of 2 per cent. Similar results after giving glu- 
cose are on record. | 

Thrown against the background of a constant basal metab- 
olism, we see outlined a definite quantitative reaction meas- 
ured in terms of extra heat production whenever protein or 
such a product of protein metabolism as glycine acts as a 
stimulus on the cells of the organism. The heat produced 
by mixing a given quantity of water and sulphuric acid 
together in a test-tube is scarcely more exactly measurable 
than are these reactions of living cells to the amino-acids or 
polypeptids which reach them after meat or kindred sub- 
stances are taken as food. 

In the light of such established facts one may investigate 
the doctrine of the specific dynamic action of protein. Rub- 
ner first showed that if the quantity of ingested protein was 
increased the heat production was increased in proportion. © 
I have shown the same of glycine. But when glycine, of 


PROBLEMS OF METABOLISM 71 


which 10 gm. alone raised the heat production of Dog 19 
20 per cent, is mixed with 40 gm. of gelatin or with the same 
amount of casein, either of which alone raised the heat pro- 
duction 30 per cent, the total increase in heat production 
remained exactly the same as though no glycine had been 
added, that is, 30 per cent. 

Here, then, is a real problem of metabolism. We remember 
the older work of Folin, how ingested glycine passes through 
the liver and may be recovered in the muscles; also the 
work of Van Slyke and of Fisher and Wishart, which showed 
that when meat itself is given amino-acids do not accumulate 
in the liver or muscles. This suggests that they are re-formed 
into protein in these localities. And now comes the recently 
published work of London! which shows that after a meal 
of meat the blood of the hepatic vein is much richer in poly- 
peptid nitrogen than is that of the portal vein. So we may 
ask ourselves, does glycine form with the broken products of 
casein a polypeptid of such a nature that it exerts no stim- 
ulating action? Or, as Professor Rubner asks after com- 
menting on this matter in a private letter to me, is poly- 
peptid nitrogen, when present in the blood, used by the cells 
to the exclusion of such an artificial product as glycine? 
So here is a problem in metabolism, something for you to 
explain if I do not find out the reason first. We have known 
about it for two years, but have found no explanation. 


METABOLISM OF FAT AND CARBOHYDRATE 


Another problem* of metabolism which has recently inter- 
ested us greatly is that of fat production in the hog. Wier- 


72 LECTURES ON NUTRITION 


zuchowski and Ling have recently repeatedly found respira- 
tory quotients above 1.5 in a young hog fed with starch and 
sugar. Even the day following the food ingestion the quo- — 
tient has been found to be 1.4. This means that a hog of 
10 kg. may readily form 100 gm. of fat daily from 270 gm. 
of starch. A daily increase of body fat of 1 per cent of the 
total body weight shows what an important fat factory the 
hog really is. When carbohydrate is converted into fat there 
is a large elimination of carbon dioxid without a correspond- 
ing demand for oxygen or a corresponding increase in heat 
production. Hence, the proportion of the volume of carbon 
dioxid eliminated to that of oxygen absorbed becomes greater 
and greater the more fat there is produced. Since the fat 
production of the hog is proportional to its respiratory quo- 
tient, we may patiently await the dawn of that scientific 
era when the price at which young pigs are sold is proportional 
to their respiratory quotients! Seriously speaking, how- 
ever, this is a problem for extensive study on the part of 
some one of our many agricultural experiment stations. An 
evident corollary of the discovery that during the fattening 
period the respiratory quotient may rise to 1.5 is that a 
calculation of the heat production from the carbon dioxid 
elimination under these conditions may lead to very gross 
errors. 

The method by which 270 gm. of cornstarch may be con- 
verted into 100 gm. of hog fat is unknown, but it is a prob- 
lem of greatest interest. It was from Hofmeister’s labora- 
tory that the suggestion came that carbohydrate lost carbon 
dioxid and produced acetaldehyde and that these molecules 


PROBLEMS OF METABOLISM PETS 


of acetaldehyde condensed, forming fatty acids of higher 
and higher order. Later Neuberg proved that acetaldehyde 
molecules were intermediary products in the alcoholic fer- 
mentation of sugar. And more recently Neuberg has pre- 
sented evidence that this highly reactive substance, acet- 
aldehyde, is also a product of mammalian metabolism. 
A. I. Ringer was the first to associate acetaldehyde with the 
mechanism of antiketosis, but Philip Shaffer finds that 
glycolaldehyde and not acetaldehyde is the effective metab- 
olite concerned in the phenomenon. The interplay among 
the broken molecules of starch from which fat is formed 
takes place at the cost of little energy. There is almost no 
waste of value here. After the living expenses of the pig 
itself have been paid for, almost all the energy of the starch 
which is taken in excess of these living expenses is recovered 
in the form of manufactured fat, created by a process which 
will some day become clear. We wish to find out all about 
this process of manufacture and have it as clear and as evi- 
dent as the manufacture of Ford cars. 

One of the manifold byways of practical application 
through scientific understanding is shown in the adminis- 
tration of glucose in cases of nervousness and prostration, 
as reported by Parker and Finley.?, From hearing of the 
symptoms of insulin hypoglycemia Mrs. Parker concluded 
that the nervous irritability and excitement manifested by 
some school children might be due to hypoglycemia. The 
reported results show that when two or three teaspoonfuls 
of glucose (commercial exose) are given in water before rising 
and also between meals in lemonade, sometimes as often as 


74 LECTURES ON NUTRITION 


six times a day, the nervous symptoms disappear and school 
work is accomplished without strain. Between 40 to 100 
gm. were given daily. In women who feel nervously fatigued 
the same course of treatment may bring about the sensation 
of vigorous well-being. During the war, when many of us 
lived on a restricted diet, lack of physical vigor may often 
have been due to hypoglycemia. Perhaps the pancakes and 
New Orleans molasses of our ancestral breakfast tables had 
their scientific justification. In England tea (containing 
sugar) is often served in the early morning before arising, 
and afternoon tea results again in raising the blood-sugar 
level. 

A certain level of blood sugar appears to be necessary for 
the proper functioning of the muscle. When the muscle 
contracts the reaction, glycogen ———— glucose ———— lactic 
acid, takes place, to be followed in the recovery phase by the 


reaction, lactic acid — glucose — glycogen, as clearly 
expounded by Hill and by Meyerhof. Doctors Deuel and 
Chambers, in our laboratory, have discovered that a dog 
weakened by the combination of long fasting and diabetes 
may be greatly restored in strength a few minutes after giv- 
ing a few grams of glucose. Correction of the hypoglycemia 
restores the muscular power even though none of the glucose 
can be oxidized by the diabetic organism. 

From this little story about glucose we can see the ines- 
capable relation between the theoretic and the practical. 
Science ever points the way; commercial prospectors gain the 
financial reward. 


In this discourse I have recited a few facts which seem to 


PROBLEMS OF METABOLISM 75 


me to be of interest and importance. It is fortunate that 
others may think differently or may entirely disapprove, 
for along such lines come discussion, experiment, and ad- 
vance in knowledge. As “der alte Voit’ used to say, “It 
matters not who is right, provided the truth becomes known.” 


BIBLIOGRAPHY 


1. London, E. S., Kotschneff, N., Kalmykoff, M. P., Schochor, N. J., and 
Abaschydze, T.: Arch. f. d. gesamt. Physiol., 1924, ccv, 482. 

2. Parker, J. T., and Finley, C. S.: Proc. Soc. Exper. Biol. and Med., 
1924, xxi, 517. 

3. Rapport, D.: Jour. Biol. Chem., 1924, Ix, 497. 

4. Rapport, D., Weiss, R., and Csonka, F. A.: Jour. Biol. Chem., 1924, 

Ee os3.) 

. Rubner, M.: Biochem. Ztschr., 1924, cxlviii, 222, 268. 

. Voit, C.: Ztschr. f. Biol., 1870, vi, 305. 

. Weiss, R., and Rapport, D.: Jour. Biol. Chem., 1924, Ix, 513. 


IHN U1 


i) 
A 


THE PROPORTIONS IN WHICH PROTEIN, FAT, AND 
CARBOHYDRATE ARE METABOLIZED IN 
DISEASE 


EvGENE F. Dv Bois 


During the last twelve years there has been a rapid growth 
in the study of the respiratory metabolism. This has been 
made possible largely through the various types of apparatus 
developed by F. G. Benedict. Clinicians have been inter- 
ested chiefly in the basal metabolism because it is an aid in 
diagnosis and treatment. They have neglected the valuable 
information furnished regarding the proportions in which 
the different foodstuffs are oxidized in health and disease. 
It is only through a careful study of these proportions that 
we can estimate the various processes which are taking 
place within our patients’ bodies. We pay a great deal of 
careless attention to the diets which we expect the patients 
to utilize, but, as Richardson” points out, we seldom have any 
idea of what really happens after the food is swallowed. Per- 
haps I should have selected the following title for this paper: 
“The food that the patient metabolizes is seldom the same as 
that which the doctor orders in the diet.” 

The human body contains large storehouses of protein and 
fat. Rubner estimates that a man weighing 73 kg. contains 
13.5 kg. of protein with its 2200 gm. of nitrogen. ‘The fat, 

77 


78 LECTURES ON NUTRITION 


of course, varies greatly according to the state of nutrition, 
but the average man contains many pounds of fat. The car- 
bohydrate stores are much smaller. To the best of our knowl- 
edge the average man stores in his liver and muscles 250 to 
400 gm. of glycogen. We may liken the body to a yacht 
with three food lockers: two large ones for protein and fat, 
and one very small one for carbohydrate. Since the crew con- 
sumes carbohydrate by preference it is obvious that the small 
locker will be emptied quickly unless the stores are replen- 
ished at frequent intervals. If they are not replenished the 
crew must get along with the protein and fat, and fortu- 
nately the stores of these are large enough to last them 
many days. These same protein and fat lockers will be drawn 
on if the steward of the yacht does not bring on board suffi- 
cient quantities of fresh food. You can see that the crew 
may subsist on a ration quite different from that which the 
steward purchases from day to day. 3 

In the case of the human body if we really want to know 
what is taking place we must review certain well-known laws 
of nutrition and, above all, adopt a somewhat different view- 
point. Perhaps the best method of doing this would be to 
start a prolonged metabolism experiment on a normal man. 
We shall assume that he is the ideal experimental subject, 
the man who can take any sort of diet without complaining. 
During our experiment we can determine his protein metab- 
olism by collecting the urine and finding its nitrogen con- 
tent. We know that each gram of urinary nitrogen in a 
given period means that 6.25 gm. of protein have been 
metabolized in about the same period. We can measure his 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 79 


respiratory metabolism and find his basal rate and his total 
calories and, what is even more important for our present 
study, we can determine his respiratory quotient. I shall not 
discuss the details of the calculation, but merely state that if 
we divide the liters of COz2 excreted by the liters of O2 con- 
sumed we can estimate the grams of fat and carbohydrate 
metabolized. In this manner it is possible to determine the 
proportions in which he actually oxidizes protein, fat, and car- 
bohydrate. Under ordinary circumstances our reckonings 
are sufficiently exact for practical purposes. We must re- 
member that there is an error of 1 or 2 per cent in all met- 
abolism calculations on account of slight differences in the 
figures used for atomic weights, factors for changing weights 
of gases to volumes, differences in caloric values, and so forth. 
There is also a certain lag in the excretion of nitrogen after 
the protein has been metabolized. The respiratory quotient 
is not reliable under changing conditions of lung ventilation, 
in acidosis, and in a few other conditions. I shall not discuss 
these possible errors at the present time, but shall leave them 
for a chapter in the mythical ‘Encyclopedia of Artifacts.” 
This important compendium, if anyone attempts its publica- 
tion, will fill many volumes, and the largest will be devoted 
to metabolism. I shall try to avoid the artifacts and base 
my discussion chiefly on the results of experiments made 
with the respiration calorimeter and other forms of apparatus 
whose reliability under the given conditions is well under- 
stood. 

In our laboratory our faith in the respiratory quotient is 
based not only on the background of theory and authority 


80 LECTURES ON NUTRITION 


but also on many forms of evidence which have been obtained 
in work with the respiration calorimeter. When alcohol is 
burned the quotient obtained corresponds closely with the 
theoretic quotient. In phlorizin diabetes and complete 
human diabetes theoretic quotients are obtained with sur- 


TEIN 
ree ee ae 


AG, 
Ace bse 
He 


Fig. 9.—Diagram showing the percentages of calories derived from 
protein, fat, and carbohydrate according to the respiratory quotient. 
The base line gives the total respiratory quotient; the ordinates reading 
on the left-hand side give the percentage of calories from protein; the 
diagonals reading on the right of the triangle give the percentage from 
carbohydrate. 


prising accuracy. In normal subjects and in patients with 
many diseases the results for the calories produced, as cal- 
culated from the respiratory quotient, correspond within 
1 or 2 per cent with the actual findings of direct calorimetry. 
Lusk’s work on the intermediary metabolism in experiments 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 81 


on dogs is based largely on the respiratory quotient, and the 
agreement with the method of direct calorimetry would be 
hard to explain if there were gross errors in the interpreta- 
tion of quotients. Perhaps one of the strongest arguments is 
the recent work of Richardson and Ladd who have checked 
the respiratory quotient against the threshold of ketosis. 
We must remember that this work was performed in hourly 
periods in a respiration chamber, but there is every evidence 
that even short periods with the technic used by Benedict, 
Boothby, Krogh, and their associates is exceedingly accu- 
rate. 

It has always been rather difficult to visualize the respira- 
tory quotient and show its significance. Fortunately the 
medical profession is now so familiar with graphic methods 
that we may resort to the use of diagrams. In Figure 9 a 
triangle has been constructed somewhat after the manner 
of the food triangles of Fisher.” ‘The base has been drawn to 
represent the respiratory quotient and the corners are as- 
signed to fat, protein, and carbohydrate." Fat has a quotient 
of 0.707, protein 0.801, and carbohydrate 1.00. Each corner 
represents the theoretic point in which all of the calories are 
derived from one of these three substances. Of course this 
never happens in real life, for we derive our calories from the 
oxidation of mixtures. The horizontal and diagonal lines 
represent the percentages derived from each. ‘The spacing 
of these lines is almost but not quite equal since the calcula- 
tion involved is rather complicated. The interpretation also 
seems complicated at first sight, but will become easier with 


the subsequent diagrams. 
6 


82 LECTURES ON NUTRITION 


In Figure 10 is shown the position of the basal metabolism 
of the normal control E. F. D. B. who happens to be the 
subject observed most often in the calorimeter. It will be 
noted that when he omits a breakfast and is studied at per- 
fect rest between 11 a.m. and 1 p.m. he derives about 19 
per cent of his calories from protein, 31 per cent from car- 
bohydrate, and the remaining 50 per cent from fat. Of course 


i 
i IN 
Ae Oe SONS 
ay aaah Pa PS NS 
JAY 7 TTP 


Fig. 10.—Dots showing the position of the basal metabolism of one normal 
man as determined in ten calorimeter experiments. 


the exact proportions depend to a large extent on his food 
of the previous day. In Figure 11 (D) are shown the effects 
of taking 200 gm. of glucose for breakfast at 10 am. The 
first calorimeter period began one hour later. At this time 
the respiratory quotient was 0.95, showing that he was de- 
riving 80 per cent of his calories from carbohydrate. ‘The 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 83 


urinary nitrogen proved that he was obtaining the other 20 
per cent from protein. For the next two hours the per- 
centages were very slightly changed, but in the fourth hour 
the quotient rose to 1.00, indicating that some of the carbo- 
hydrate was being transformed into fat. 


4 


[so 
aia 


0 
Fig. 11.—Triangle used to show changes in metabolism: R, the effect 


of a large protein meal; D, the effect of 200 gm. of glucose; L, the first 
six days of starvation. 


On another day a moderate sized protein meal containing 
10.5 gm. of nitrogen caused the protein metabolism to in- 
crease until in the fifth hour it furnished 29 per cent of the 
calories, carbohydrate contributing 25 per cent, and fat the 
remaining 46 per cent. This test is not illustrated in Figure 
11. Much more striking results were obtained in the case of 
an achondroplastic dwarf! selected because his arms and 


84 LECTURES ON NUTRITION 


legs were very small and his appetite and stomach very large. 
This little man who weighed but 90 pounds ate chopped meat 
steadily for one hour and consumed a total of 662 gm. of 
beef with its 23.2 gm. of nitrogen. In other words, he took 
for breakfast about twice as much protein as the ordinary 
man eats in one day. The calorimeter period which began 
one hour after this unusual meal is shown in Figure 11 (R). At 


SIEEe un 


MEU Eber 
HY pee EERE 
in SP AoMor ENGLER 


' Fig. 12.—Zones of metabolism. The dots under the R. Q. line show 
the lowest respiratory quotients obtained in severe diabetes. The dots 
under the D : N line show the highest D : N ratios found in the literature. 


this time he was obtaining 47 per cent of his calories from 
protein. Between the fourth and fifth hours he was deriving 
64 per cent from this source and the quotient of 0.77 indi- 
cated that fat was supplying 34 per cent. 

In Figure 12 there have been added certain zones outside 
of the triangle. When carbohydrate which contains a large - 
proportion of oxygen is transformed into fat with little 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 85 


oxygen the respiratory quotient rises above unity, sometimes 
to 1.2 or even 1.5. The zone of this transformation lies, there- 
fore, to the right of the triangle. To the left of the triangle 
is found another zone which is never invaded by normal 
men. We know that 100 gm. of protein can furnish in the 
metabolism of the living body about 58 gm. of glucose. In 
severe diabetes this glucose is excreted in the urine and the 
respiratory quotient falls below 0.707. Since protein rarely 
furnishes more than 30 to 40 per cent of the total metab- 
olism we seldom approach the upper portions of this zone 
or reach the lowest theoretic quotient, 0.63. 

There are probably a number of chemists in this audience 
who are looking for the zone in which fat is transformed 
into carbohydrate. If this really took place the area would 
lie to the left of the main triangle and would extend far to 
the left. We would obtain many respiratory quotients be- 
low 0.65, especially in cases of severe diabetes. It is quite 
easy for the chemist to see how glycerol would be changed 
into glucose and there is much evidence to prove that this 
actually occurs, but glycerol forms such a small part of 
the fat molecule and the quotient is so little affected that the 
process cannot be detected in the quotient. The chemist 
can also write reactions showing the theoretic transforma- 
tion of fatty acid into glucose, and most investigators in 
Europe believe that this actually occurs. Personally, I am 
quite convinced by the arguments of Lusk and other Ameri- 
can biochemists that this has never been proved. If it did 
occur, all the modern theories of antiketogenesis would fall 
by the board. If fat were changed into glucose, this glucose 


86 LECTURES ON | NUTRITION 


would be excreted in cases of complete diabetes, giving us 
many respiratory quotients below 0.65 and many D : N 
ratios above 3.65. In Figure 12 I have plotted the most 
reliable data which could be found in the literature. You 
will note that few are beyond the limits mentioned and 
these slight discrepancies could easily be due to experi- 
mental error. This means that the complete diabetic ex- 
cretes no sugar that does not come from carbohydrate or the 
carbohydrate portion of the protein molecule or from gly- 
cerol. It also means that he does not transform an oxygen- 
poor substance like fatty acid into an oxygen-rich sub- 
stance like carbohydrate. We can only use the condition of 
complete diabetes to make these deductions, because all 
normal men have carbohydrate available for oxidation and 
we cannot prove that it was not derived from fat. It is quite 
possible that fat passes through some carbohydrate-like stage 
as it is oxidized. All we can say is that it passes through 
this stage so quickly that it makes no difference to the prac- 
tical results. We may illustrate this point by returning to 
the yacht with its three food lockers. If the crew called for 
carbohydrate and the carbohydrate locker were empty the 
steward might take some fat and place it for an instant in 
the carbohydrate locker before serving it. He might call it 
carbohydrate because it had been in that locker, but for all 
practical purposes it would remain fat. 

Having thus explored the territory which surrounds the 
triangle it is necessary to return to the affairs of every-day 
life and study the factors which affect the position of the — 
metabolism within the triangle. In a previous lecture of 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 87 


this series Hill * has shown, by means of his brilliant experi- 
ments, that in periods of short violent exercise the energy 
is derived solely through the oxidation of carbohydrate. He 
has emphasized the fact that respiratory quotients are falsely 
high during exercise and falsely low during recovery so that 
we must average the COz and Oz of a long series of con- 
secutive periods which cover both phases. He has shown 
that we can subtract from such a work period the basal CO, 
and O. and find the quotient of the work itself, together with 
its recovery phase. Protein seems to play but little part as 
a source of energy for muscular work. This was shown long 
ago by Voit and confirmed in the classical experiment of 
Fick and Wislicenus,” who found no increase in the nitrogen _ 
excretion when they climbed a high mountain in Switzerland. 
Kocher has shown the same thing even more clearly by 
placing himself on a nitrogen minimal excretion of 2.9 gm. 
a day, obtaining a negligible increase on the day when he 
walked 60 kilometers. Thomas” has obtained similar results. 
On the other hand, it is quite evident that protein can fur- 
nish a considerable portion of the energy for muscular work 
in carniverous animals. It is also evident that fat must 
furnish the chief source of muscular energy in the case of 
individuals like the Eskimos who consume practically no 
carbohydrate yet are capable of great feats of exertion. 
It is estimated! that some of the Eskimos derive only 8 per 
cent of their calories from carbohydrate since they consume 
only 50 or 60 gm. a day. This is obviously too small an 
amount to carry them on long hunting expeditions. In 
respiration experiments performed on individuals who are 


88 LECTURES ON NUTRITION 


taking moderate or long-continued exercise there is sur- 
prisingly little change in the quotient when we discount the 
sudden driving off of carbon dioxid due to the lactic acid 
formation and suddenly increased pulmonary ventilation. 

In New York our interest in this phase of the subject 
was greatly stimulated by A. V. Hill’s visit to this country, 
and we immediately recalculated all of our calorimeter ex- 
periments in which the subjects had performed mild amounts 
of exercise such as might readily be accomplished by hos- 
pital patients. We also had at our command a certain num- 
ber of observations made by Barr, Cecil, and Du Bois,? in 
which the patients had shivered quite violently during chills 
caused by malarial paroxysms or by the intravenous injec- 
tion of foreign protein. In all of these experiments the sub- 
jects were observed for more than an hour after the muscular 
work so that we could obtain the phase of recovery during 
which the excess of lactic acid was reconverted into gly- 
cogen. It was, therefore, possible to find the respiratory 
quotient of the work increment. Doctors Richardson and 
Levene have kindly allowed me to add calculations taken 
from their article on exercise in diabetes which is as yet un- 
published. It will be noted that there is little difference 
between the basal quotients and those of the exercise incre- 
ment, the average for the former being 0.835, for the latter 
0.782. This means that these patients during mild exercise 
consumed fat and carbohydrate in about the same propor- 
tions as when they were resting. 

At the beginning of this lecture I emphasized the fact 
that the diet on any given day might differ greatly from the 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 89 


TABLE 1 


EFFECT OF MILD EXERCISE ON R. Q. 


Subject Exercise Basal Exercise 
R.Q. Increment 
E,F.D.B,. Normal Shivering 34 min, 0.83 0.86 
E.F.D.B. u oe 35 i 0.81 0.87 
Chas. R. Congenital ® 34 0 0.88 0.63 
absence of ‘ 
sweat giands 
Gsorge 5. Malarial ohill " 34 40 0.78 0.83 
Joseph MoC. Typhoid vac. " " 30 «9 0,838 0.72 
0 f ? " Ld " 33 a 0,89 0.73 


Average 0,835 Average 0.783 


David L. Diabetic Mild exeroise 40 min, 0.80 0.61 
Morris G, Ae a a " 59 * 0.77. 0.74 
Ray #,. * ® " ey 0.85 0.81 
Jervis B. ) ® ” 97° * 2 0.83 0.72 
et itd ° ® ® 63 * 0.80 0.73 
Nicholas &, ® 2 My 36 0.80 0.73 
James D, * ms Sone 0,80 0.77 
Gerald 8. be! ~ 2 10 ~«(* 0.76 0.84 
Average diabetic 0.80 0.768 


food actually metabolized. Conditions, however, are en- 
tirely different in normal individuals subsisting for months 
and years on any fairly uniform dietary which is sufficient 
to supply the bodily needs and prevent loss of weight. In 
the course of a year the average metabolism of the race 
must correspond quite closely with the average composition 
of the diet. We can, therefore, study with profit the stand- 
ard dietaries of different races, as shown in Figure 13. It is 
interesting to note that the Eskimo takes five times the 
amount of protein eaten by the Bengali. We must, of course, 
remember that the position of the metabolism on such a 


90 LECTURES ON NUTRITION 


diagram changes after each meal and that the points shown 
are merely averages. 

On this chart there are certain zones which are not ordi- 
narily reached in daily life. Of particular interest is the 
zone of low protein metabolism such as can be obtained in 
experiments on the nitrogen minimum. Most Americans 


CAR B. 


bs 


Fig. 13.—Position of various racial dietaries: E, Voit’s European “‘Stand- 
ard.’ B, Bengali. M, Maine lumberman. 5S, Eskimo. 


under ordinary conditions derive about 15 to 20 per cent 
of their calories from protein, some as little as 10 per 
cent. Since the protein metabolism does not increase with 
muscular exercise, it is obvious that a man who runs a race, 
increasing his total metabolism ten times or more, will, dur- 
ing the period of muscular exertion, derive only 1 or 2 per 
cent of his calories from protein. A great deal of informa- 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 91 


tion has been furnished by observations on the protein mini- 
mum. Klemperer, Sivén, Landergren, and others gave men 
an ample diet, rich in carbohydrate, containing 2 to 2.5 gm. 
food nitrogen. On this diet the men excreted 3 to 4 gm. of 
nitrogen daily. ‘Thomas obtained even lower results and 
actually placed himself on nitrogen balance at the very low 
level of 2.2 gm. of nitrogen a day. Thomas’ experiments 


AVRO WIIG ISAT MONA 23) eS. 


Fig. 14.—Experiment of Thomas on the nitrogen minimum. The con- 
tinued line shows the urinary nitrogen. The dotted line represents food 
nitrogen. 


are illustrated in Figure 14. His work demonstrates the 
fact that carbohydrates are more efficient than fats as sparers 
of protein. It also proves that we can give small amounts of 
protein in a diet without increasing the protein metabolism. 
Altogether, his long experiment is the best demonstration of 
the “‘wear and tear quota” of Rubner, that minimal breaking 
down of protein which seems to be necessary for life. 
Having considered the effects of foods we must next take 


92 LECTURES ON NUTRITION 


up the factor of undernutrition since it enters into the pic- 
ture of almost all patients who are seriously il. The most 
complete experiment that has ever been made in starvation 
was performed by Benedict* and his associates. They had 
the unusual opportunity of studying the subject Levanzin 


BENEDICT S SUBJECT L” 


is 4 


x \2 
ROS OPPO 
q SJ 
SEBO etek ecete ate Se 


SSPOL oa % 4 
SES < 


SOR 
SRR 
ROSS esos 
SHS Rox RSI 
Soto Shs SSIS 
Sout Sy RRR SSE oes 


Fig. 15.—Benedict’s fasting subject, Levanzin. Materials oxidized during 
first fourteen days of starvation. 


under almost ideal conditions since he slept in the respira- 
tion calorimeter every night during his thirty-one-day fast. 
Additional respiration experiments were made in the day- 
time. The results of the first fourteen days are shown in 
Figure 15. It will be noted first that there was a gradual fall 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 93 


in the total heat production. The amount of carbohydrate 
oxidized was quite considerable on the first day but de- 
creased rapidly as the glycogen stores were depleted, until 
after the fifth day only traces were consumed. 

The protein metabolism was comparatively low in the 
first day of the fast and he only excreted 7.1 gm. of nitrogen 
in the urine. When the carbohydrate decreased the pro- 
tective effect of this substance on the protein destruction 
was diminished to such an extent that the nitrogen elimina- 
tion rose to 11.9 gm. Later it fell to the level of about 8 
gm. a day, partly on account of the diminished total metab- 
olism, partly perhaps because the organism was adapting 
itself to the new conditions. It will be noted in the chart 
that the chief source of heat was the fat derived from his 
rather ample stores, although Levanzin was by no means 
obese at the beginning of his fast. 

In this particular subject Benedict had the unusual oppor- 
tunity of studying the amount of glycogen which had been 
stored in the liver and muscles at the beginning of the fast. 
The muscles contain about 0.3 per cent of glycogen, but 
the liver is the chief storehouse since its glycogen content 
may be very high. A dog’s liver may hold 18.7 per cent of 
its weight as glycogen and there is no reason to suppose that 
this level cannot be reached in man. The calculation of the 
total carbohydrate storage in man is made by noting the 
percentage of calories furnished by carbohydrate during the 
respiration experiments and assuming that this percentage 
holds good for the periods when the subject is outside the 
calorimeter. In Levanzin’s case Benedict found that 201 


/ 


94 LECTURES ON NUTRITION . 


gm. of glycogen were available. It is doubtful if we shall 
ever know how completely the glycogen stores are exhausted 
in the starving human subject. Experiments on phlorizin- 
ized dogs by Lusk and others have shown that it is neces- 
sary to use not only prolonged fasting but also adrenalin 
and periods of shivering or other violent exercise which 
mobilize the glycogen so that it is available for excretion by 
the kidneys. Richardson and Ladd,” in their observations 
on human diabetes, have obtained indications that there 
may still be considerable amounts of glycogen stored in the 
body, even after periods when there has been little or no 
evidence of carbohydrate metabolism. 

Complete starvation is not frequently encountered in the 
clinic, but partial undernutrition is almost universal in pa- 
tients who are seriously ill. Normal men on inadequate 
diets show a gradual fall in the basal metabolism and, as a 
rule, a rather low nitrogen output. The materials that they 
actually consume in their bodies depend largely on the food 
that is administered. If they are given protein alone, the 
nitrogen excretion will rise to a point somewhat above the 
nitrogen intake and they will never attain nitrogen equilib- 
rium. ‘This was shown in the experiments of Thomas.” If 
they are given fat alone, there will be a sparing of body fat 
but a depletion of the stores of carbohydrate and protein. 
If they are given carbohydrate alone, the fat stores will be 
protected and if the caloric intake is equal to the total heat 
production, there may be an almost complete protection of 
the fat. Under such conditions the protein metabolism will 
be greatly diminished and the nitrogen minimum attained, 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 95 


as in the experiments of Thomas. If carbohydrate alone is 
given, but in amounts which do not cover the total require- 
ment, the sparing of fat and protein will, of course, be much 
less marked. As a rule, undernourished subjects are given 
mixtures of food, and the level of the carbohydrate and pro- 
tein metabolism will depend on the amounts of these sub- 
stances ingested. In all cases of undernutrition fat must 
furnish the rest of the calories and this fat is obtained partly 
from the food but chiefly from fat which has been previously 
stored in the body. 

Let us return once more to the simile of the yacht with 
its three food lockers. If this yacht sails on a long voyage 
without touching port, the carbohydrate locker will be almost 
emptied in the first few days. The protein locker will be 
drawn on moderately each day and the bulk of the food 
will come from the fat stores. If the yacht does touch port 
at more or less regular intervals and the steward is not able 
to secure sufficient and proper rations, the crew will get along 
as best it can, utilizing completely the fresh supplies, what- 
ever they might be, but drawing when necessary upon the 
stores which filled the lockers at the beginning of the 
voyage. 

We must next consider the proportions in which fat and 
carbohydrate are oxidized and their relationship to ketosis. 
The subject has been made so familiar to you by the recent 
brilliant work of Woodyatt*» * and Shaffer?’ that I shall not 
try to discuss the chemical aspects. Rosenfeld, in 1885, said, 
“The fats burn in the fire of carbohydrate.” It has long been 
known that the complete oxidation of fat in the body could 


96 LECTURES ON NUTRITION 


not be obtained unless some carbohydrate were being oxidized 
at the same time. If fat is not completely oxidized, aceto- 
acetic and beta-hydroxybutyric acids are left in the tissues, 
causing the condition known clinically as ketosis. Carbo- 
hydrates are called antiketogenic because in their oxidation 
they prevent the formation of the ketones or aid in their 
complete combustion. Woodyatt,* in 1910, suggested that 
1 molecule of aceto-acetic acid reacted with 1 molecule of 
an alcohol or glucose. The experimental work and calcula- 
tions of Shaffer and of Woodyatt® have confirmed this 
relationship, although there is some evidence!” !® * that 1 
molecule of glucose may be able to take care of more than 
1 molecule of fatty acid. When the available carbohydrate 
is insufficient the organism ‘‘smokes”’ like a gasoline engine 
with an improper mixture. 

It is possible to indicate this zone of ketosis on the tri- 
angle, and Figure 12 shows the line where ketosis appears 
as the carbohydrate metabolism is diminished. This line 
corresponds to the equimolecular ratio of Shaffer and Wood- 
yatt’s fatty acid-glucose ratio of 1.5. At the right hand 
border of this zone the ketosis is mild. Near the extreme fat 
corner it is severe. The starving man Levanzin was within 
this zone after the first day of his fast. The highest degrees 
of ketosis are, of course, found in diabetes. 

It seems possible at the present time to ascribe all the 
various phenomena of diabetes to a diminution in the secre- 
tion of insulin by the pancreas. In mild cases, in which 
the loss of function is slight, the metabolism is not very 
different from that of normal men. In cases of moderately 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 97 


severe diabetes the fat metabolism must predominate and 
only a small percentage of the calories is derived from car- 
bohydrate. In cases of severe diabetes the protein metab- 
olism is affected, since the diabetic organism can no longer 
utilize the carbohydrate portion of the protein molecule. 
The patient with so-called ‘‘complete diabetes” excretes 58 
gm. of glucose for each 100 gm. of protein metabolized. He 
can oxidize practically no glucose from any source to aid in 
the metabolism of the fatty acids. As a result the forma- 
tion of aceto-acetic and betahydroxybutyric acids is enor- 
mous. | 

The symptoms of severe ketosis are well known: deep res- 
piration, weakness, nausea, somnolence, coma. The symp- 
toms of mild ketosis are much less marked, but diabetics and 
normal men on a low carbohydrate diet show weakness, 
lassitude, inability to perform much mental or physical 
labor, and lack of resistance to infection. JI am inclined to 
believe that there may be a distinctly lowered resistance 
to the effects of disease in the zone where the organism just 
escapes a ketosis, but I do not know of any exact data on 
this point. 

The diabetic organism exists chiefly on fat. If the pa- 
tient is starved he lives on body fat. His fat metabolism is 
not necessarily increased by giving him fat in his food. This 
was shown by Richardson and Mason” (Fig. 16). The fat 
metabolism can, however, be reduced by means of fasting 
which reduces the total metabolism. If the carbohydrate 
tolerance remains fixed this improves the fatty acid-glucose 


ratio. Nowadays it is possible to increase the carbohydrate 
7 


98 LECTURES ON NUTRITION 


tolerance by means of insulin and this gives us even a better 
means of improving the ratio and avoiding ketosis. 

Obese patients are often placed on very low diets in order 
to get rid of the surplus fat. In such cases it is necessary to 


CAL Pot 
2000 JAMES F 


FRANCIS M. CHAS .C.—_—y 


OP OP ey ey ie eet 
4500 


|__|} ee 
ill | 
‘ 
X 
2 


. 
° 
9 


IE 
vp) 
< 
we 


®= CALORIES IN DIET. $=CALORIES, METABOLIZED, 


BEN.J — FRANK C.—~—PAT, Maat FRANK B.—s 


en Poyog. Popeye yey of fy 


ih 
J 
: 
ii if : 


Fig. 16.—Observations of Richardson and Mason on the effect of food 
in diabetes. The columns under the circles represent the food given in 
the diet. The columns under the arrows represent the materials actually 
metabolized. The cross-hatched areas are the protein calories, the ver- 
tical lines represent the carbohydrate calories. Fat fed is shown in solid 
black, fat metabolized, in solid white. 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 99 


estimate the total metabolism and see that enough carbo- 
hydrate calories are supplied to balance the large metabolism 
of fat. 

Our diets are voluntarily restricted in obesity, but are re- 
stricted by stern necessity in almost all patients who are 
seriously ill. Loss of appetite, repugnance for food, nausea, 
and vomiting force the doctor to resort to low diets. Some- 
times he is quite content to give the stomach a rest for a 
few days. ‘This, however, does not give the metabolism a 
rest. The metabolic picture is just the same as in the starv- 
ing man, Levanzin. There is a slight fall in total metab- 
olism, and a rapid exhaustion of the glycogen stored in 
muscles and liver. The organism passes into the zone of 
ketosis and the evil effects of starvation are added to those 
of the disease. 

Fortunately, it is almost always possible for skilful nurses 
to give some food. The most important food is carbohydrate 
because this will replenish the glycogen stores and tend to 
diminish destruction of body protein. Protein food is of 
next importance. Fat comes last of all because the human 
body has large enough stores to last for many days, and if 
these are depleted during illness they can be refilled during 
convalescence. 

Experienced physicians have made a consistent effort in 
recent years to administer carbohydrate by mouth or glucose 
by vein or rectum. Even small amounts of glucose may be 
of great service. In diabetes we struggle hard to secure the 
metabolism of each additional 10 gm. of carbohydrate. 
When the patient who has been on a diet of 20 gm. increases 


100 LECTURES ON NUTRITION 


his tolerance so that he can take 30 gm. there is great re- 
joicing on the part of the physician. In a medical or surgical 
patient on desperately low rations we should likewise rejoice 
over each additional 10 gm. that is retained within the body. 
If it is once absorbed into the blood it will be metabolized. 
As I have said before, the factor of undernutrition plays 
an important part in the metabolic picture of every patient 
who is seriously ill. There are, of course, many other factors 
which are part and parcel of the particular disease in ques- 
tion. By all odds the most important is infection with its 
resulting fever. Fever raises the basal metabolism in pro- 
portion to the rise in temperature.!° It seems to cause no 
particular change in the carbohydrate and fat metabolism. 
Carbohydrates are oxidized if available; if not available, fat 
supplies the necessary heat. The protein metabolism, how- 
ever, is distinctly increased in all infections which are accom- 
panied by that somewhat indefinite condition known as tox- 
emia. Here we encounter the so-called toxic destruction of 
protein. The patient cannot be brought into nitrogen equi- 
librium even though we give in the diet enough calories to 
cover the total heat production. ‘This phenomenon was 
carefully studied in 1909 by Shaffer and Coleman. ‘They 
administered to typhoid patients diets containing 3,000 or 
4,000 calories largely in the form of carbohydrate. In doing 
this they eliminated from the clinical picture of typhoid 
fever the usual factor of partial starvation and, incidentally, 
they showed that most of the distressing features of typhoid 
were due to starvation rather than infection. By means of 
these large diets they were able to bring a few patients into 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 101 


nitrogen equilibrium. The subsequent studies by Coleman 
and Du Bois’ showed that these diets contained almost twice 
as many calories as the patients actually produced. It is 
quite obvious that there must have been some abnormal 
factor increasing the protein metabolism. Shaffer and Cole- 


APY! 
Ariba 
VU 


LOW CALORY Del-tacd 


Fig. 17.—Metabolism map of typhoid patients on low calory diet: A, 
Ascending temperature period; C, continued temperature; E, early steep 
curve; L, late steep curve; 1, first week of convalescence; 2, second week 
of convalescence, etc. 


man also tried to ascertain the nitrogen minimum in typhoid 
fever and found that it was impossible even on an ample diet 
containing little protein to bring down the nitrogen elimina- 
tion to the low levels obtained by Landergren and others in 
health. Kocher confirmed this in paratyphoid fever. In 
Figure 17 I have sketched the position of the basal metab- 


102 LECTURES ON NUTRITION 


olism during the different weeks of typhoid fever as estimated 
from the respiration experiments recorded in the German 
literature before 1912. These patients were given the old- 
fashioned restricted fever diet, and the respiratory quotients 
were measured twelve hours or more after the last meal. 
Carbohydrate furnished a very low percentage of calories 


er ereity Donal 


Fig. 18.—Typhoid patients on high calory diet. 


during the fever. In convalescence, when the patient was 
consuming enormous amounts of food, the carbohydrate 
plethora lasted so long after the evening meal that little or 
no fat was metabolized. 3 

Figure 18 shows the results obtained in the patients who 
were placed on the Coleman-Shaffer high calory diet. Dur- © 
ing the first part of the fever this could be administered in 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 103 


large amounts, but later, as the patients’ appetites improved, 
they were brought into about the same metabolic position 
that is occupied by normal men. The toxic destruction of 
protein was checked though not abolished, the patients were 
removed safely from the zone of ketosis, and the metabolic 
phenomena of convalescence were often clearly manifest 
several weeks before the febrile period ended. Some of the 
patients even with high temperatures were able to replenish 
the stores of body fat. This diet has changed the clinical 
picture of typhoid fever and caused a distinct reduction in 
the mortality. 

No other fever has been studied as thoroughly as typhoid. 
From the limited data on hand, however, we can say that the 
picture is almost exactly duplicated in erysipelas and prob- 
ably the other fevers with similar toxemia. Tuberculosis is 
a much more chronic disease than typhoid and the toxemia 
does not seem to be so violent. McCann and Barr” have shown 
that the normal levels of nitrogen minimum can be rather 
closely approximated in tuberculosis, thus proving that 
there is much less toxic destruction of protein in this disease. 
If time would permit I should like to discuss the experi- 
ments of McCann” on the effect of food on the pulmonary 
ventilation in tuberculosis. He proved that if much pro- 
tein were given, the specific dynamic action would increase 
the respiratory exchanges and cause harmful increments in 
the work of the lungs. He pointed out the dangers of over- 
feeding and showed that the metabolism of a given number 
of calories in the form of fat was accompanied by a dis- 
tinctly lower pulmonary ventilation than if a diet which con- 


104 LECTURES ON NUTRITION 


tained the same number of calories in the form of carbo- 
hydrate were administered. 

So much for the factors of fever and infection. We must 
next turn our attention to the circulation and the kidneys. 
It is doubtful if we shall ever be able to find out the true 
level of protein metabolism in heart failure or severe renal 
disease. In these two conditions the urinary output is so 
irregular that it gives us no indication of the rate of protein 
metabolism. All we can do is to assume that the protein 
metabolism will be at least as high as in starving men. If 
this is the case, we can give cardiac and nephritic patients 
at least 4 to 6 gm. of nitrogen in the food without causing 
the slightest increase in the formation of urea. There does 
not seem to be any valid reason for the protein-free diets 
that have sometimes been advocated. If we really want to 
depress the formation of urea to the lowest possible point in 
kidney disease we should give a diet of ample calories con- 
taining 20 to 30 gm. of protein. We must, of course, take 
into consideration all the other factors of the disease, paying 
due attention to the gastro-intestinal tract, and so forth. 
In heart failure and in some cases of nephritis many clinicians 
employ with excellent results the diet of 1 liter of milk with- 
out any additional fluids. This is a submaintenance diet and 
the patient is, therefore, losing some of his body fat. 

In hyperthyroidism the increase in total metabolism over- 
shadows all the other metabolic phenomena. It has long 
been recognized that if we wish to keep these patients in 
nitrogen balance we must give a large excess of calories 
above the basal. Boothby and Sandiford® have recently 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 105 


explained the reason for this apparent excess. They have 
shown that in hyperthyroidism a given amount of work 
requires about twice the expenditure of calories that it does 
in normal individuals. They have found no evidence of 
abnormal protein metabolism. Rudinger” in studying two 
young patients demonstrated only a slight increase in the 
nitrogen minimum. More experiments on this minimum 
are needed badly. The carbohydrate metabolism in hyper- 
thyroidism is supposed to be abnormal, but I am not familiar 
with any definite proof of such abnormality. One of our 
patients? in the Sage calorimeter, in 1914, showed a gly- 
cosuria, yet after taking 100 gm. of glucose was able to 
derive 76 per cent of his calories from this source, his res- 
piratory quotient being 0.943. Sanger and Hun” have re- 
cently noted the same phenomenon. Cramer,’ as a result of 
his work on rats, advanced the hypothesis that in hyper- 
thyroidism the liver could not store glycogen. In 1916 we 
had an unusual opportunity of studying glycogen storage in 
a young man with exophthalmic goiter who fasted for three 
days during which time he was observed at intervals in the 
calorimeter. His respiratory quotient fell at almost exactly 
the same rate as Benedict’s fasting subject, Levanzin. It 
was possible to calculate that he oxidized in the first three 
days between 300 and 360 gm. of carbohydrate, which is 
well within the normal limits. This result has been con- 
firmed recently by Richardson and Levene. 

The studies of recent years have been of great service in 
removing many of the mysteries that enveloped the metab- 
olism in disease. Our subject has become much simpler than 


106 


LECTURES ON NUTRITION 


it was ten years ago. We have reached the point where by 


means of diet we can usually modify the patient’s metabolism 


and accomplish certain specific results. These results, how- 


ever, can be attained only by careful study of the various 


processes which are taking place within the patient’s body. 


BIBLIOGRAPHY 


. Aub, J. C., and Du Bois, E. F.: Clinical Calorimetry Paper 21. The 


Basal Metabolism of Dwarfs and Legless Men with Observations on 
the Specific Dynamic Action of Protein, Arch. Int. Med., 1917, 19, 
842-864. 


. Barr, D. P., Cecil, R. L., and Du Bois, E. F.: Clinical Calorimetry 


Paper 32. Temperature Regulation After the Intravenous Injection 
of Proteose and Typhoid Vaccine, Arch. Int. Med., 1922, 29, 608-634. 


. Barr, D. P., and Du Bois, E. F.: Clinical Calorimetry Paper 28. The 


Metabolism in Malarial Fever, Arch. Int. Med., 1918, 21, 627-658. 


. Benedict, F. G.: A Study of Prolonged Fasting, Carnegie Institution 


of Washington Pub. No. 203, 1915. 


. Boothby, W. M., and Sandiford, I.: Total and Nitrogenous Metabolism 


in Exophthalmic Goiter, Jour. Amer. Med. Assn., 1923, 81, 795-800. 


. Coleman, W., and Du Bois, E. F.: The Influence of the High Calory 


Diet on the Respiratory Exchange in Typhoid Fever, Arch. Int. 
Med., 1914, 14, 168. 


. Coleman, W., and Du Bois, E. F.: Clinical Calorimetry Paper 7. 


Calorimetric Observations on the Metabolism of Typhoid Patients 
With and Without Food, Ibid., 1915, 15, 887-938. 


. Cramer and Krause: Proc. Royal Soc. B., 1913, 86, 550. 
. Du Bois, E. F.: Clinical Calorimetry Paper 14. Metabolism in Exoph- 


thalmic Goiter, Arch. Int. Med., 1916, 17, 915-964. 


. Du Bois, E. F.: The Basal Metabolism in Fever, Jour. Amer. Med. 


Assn., 1921, 77, 352-357. 


. Du Bois, E. F.: Clinical Calorimetry Paper 35. A Graphic Repre- 


sentation of the Respiratory Quotient and the Percentage of Calories 
from Protein, Fat, and Carbohydrate, Jour. Biol. Chem., 1924, 59, 
43-49, 


. Fick and Wislicenus: Myothermische Untersuchungen, 1889. 
. Fisher, I.: Amer. Jour. Physiol., 1906, 15, 417; Jour. Amer. Med. 


Assn., 1907, 48, 1316. 


. Hill, A. V.: Muscular Activity and Carbohydrate Metabolism, Science, 


1924, 60, 505-514. 


15. 


16. 


17, 


18. 
19. 


20. 


21. 


Dia 


ZS. 


24. 


25: 
26. 
dale 
28. 
29. 
30. 


31. 


32. 


PROTEIN, FAT, AND CARBOHYDRATE IN DISEASE 107 


Hill, A. V., Long, C. N. H., and Lupton, H.: Muscular Exercise, Lactic 
Acid, and the Supply and Utilization of Oxygen, Parts iv to vi, Proc. 
of the Royal Soc. B., 1924, 97, 84-138. 

Kocher, R. A.: Ueber die Grosse des Eiweisszerfalls bei Fieber und 
bei Arbeitsleistung, Deutsch. Arch. f. klin. Med., 1914, 115, 82. 

Ladd, W. S., and Palmer, W. W.: The Carbohydrate-fat Ratio in 
Relation to the Production of Ketone Bodies in Diabetes Mellitus, 
Proc. Soc. Exper. Biol. and Med., 1920-21, 18, 109. 

Ladd, W.S.,and Palmer, W. W.: The Useof Fat in Diabetes Mellitus and 
the Carbohydrate-fat Ratio, Amer. Jour. Med. Sci., 1923, 166, 157. 

Lusk, G.: The Fundamental Basis of Nutrition, New Haven, Yale 
Univ. Press, 1923, 

McCann, W. S.: The Effect of the Ingestion of Foodstuffs on the 
Respiratory Exchange in Pulmonary Tuberculosis, Arch. Int. Med., 
1921, 28, 847-858. 

McCann, W. S., and Barr, D. P.: Clinical Calorimetry Paper 29. The 
Metabolism in Tuberculosis, Arch. Int. Med., 1920, 26, 663-705. 
Richardson, H. B.: The Inadequacy of the Measured Diet as an Index 
of the Food Metabolized, Boston Med. and Surg. Jour., 1923, 189, 

813-819. 

Richardson, H. B., and Ladd, W.S.: Clinical Calorimetry Paper 34. 
Ketosis and the Respiratory Exchange in Diabetes, Jour. Biol. 
Chem., 1924, 58, 931-968. 

Richardson, H. B., and Mason, E. H.: Clinical Calorimetry Paper 33. 
The Effect of Fasting in Diabetes as Compared with a Diet Designed 
to replace the Foodstuffs Oxidized During a Fast, Jour. Biol. Chem., 
1923, 57, 587-611. 

Rudinger: Ueber den Eiweissumsatz bei Morbus Basedowii, Wien. klin. 
Wchnschr., 1908, 21, 1581. 

Sanger, B. J., and Hun, E. S.: The Glucose Mobilization Rate in 
Hyperthyroidism, Arch. Int. Med., 1922, 30, 397-406. 

Shaffer, P. A.: Antiketogenesis, Jour. Biol. Chem., 1921, 47, 433, 449; 
Ibid., 49, 143; Ibid., 1922, 54, 399. 

Shaffer, P. A., and Coleman, W.: Protein Metabolism in Typhoid 
Fever, Arch. Int. Med., 1909, 4, 538-600. 

Thomas, K.: Ueber das physiologische Stickstoffminimum: Arch. f. 
Anat. u. Physiol., 1910, Supplement Bd., 249-285. 

Wilder, R. M., and Winter, M. D.: The Threshold of Ketogenesis, 
Jour. Biol. Chem., 1922, 52, 393. 

Woodyatt, R. T.: The Action of Glycolaldehyd and Glycerin Aldehyd 
in Diabetes Mellitus and the Nature of Antiketogenesis, Jour. Amer. 
Med. Assn., 1910, 55, 2109. 

Woodyatt, R. T.: Objects and Method of Diet Adjustment in Diabetes, 
Arch. Int. Med., 1921, 28, 125. 


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MUSCULAR ACTIVITY AND CARBOHYDRATE 
METABOLISM 


ARCHIBALD VIVIAN HILL 


THE PROBLEM: INTRODUCTION 


It has long been discussed whether the breakdown of 
carbohydrate, rather than of other substances, is primarily 
responsible for the provision of energy in muscular con- 
traction. It is known and accepted that work may be done, 
in the general melting-pot of the body, by the use of any 
kind of foodstuff. We are now concerned, however, specif- 
ically with the primary process of muscular contraction. In 
the complete chain of processes involved in long-continued 
exercise, this primary process may be disguised, or even 
apparently obliterated, by simultaneous transformations 
which take place between the different food constituents. 
Considering the internal combustion engine, it is obvious 
that petrol and benzole may be used indiscriminately for 
providing power and driving the machinery. In the same 
way, however, as. we ask whether carbohydrate is the specific 
fuel of muscle, or whether fat may be used in an identical 
manner, so we might query whether petrol or coal can be 
used in an internal combustion engine. The obvious answer 
is that coal must be prepared beforehand by distillation, 


before it can be used in the engine, while petrol can be used 
109 


110 LECTURES ON NUTRITION 


directly; and that in the preparation of coal to form benzole 
for use in the engine, a considerable proportion of the energy 
of the coal is wasted, as regards its work-producing power. 
Putting our problem in terms of the modern theory of mus- 
cular activity and assuming that the initial process in con- 
traction—that which causes the mechanical response—is an 
entirely non-oxidative one, consisting of the formation of 
lactic acid from glycogen, we are asking now whether the 
recovery process by which the lactic acid is restored to its 
precursor can go on at the expense of any oxidation, or only 
of that of carbohydrate. May the recovery mechanism, so 
to speak, be driven by any kind of combustion, as a steam engine 
may be, or 1s it necessary specifically to combust carbohydrate? 


THE RESPIRATORY QUOTIENT 

It has long been known that the respiratory quotient 
during prolonged steady exercise is not unity. It varies with 
the diet. That, however, does not answer our question. 
Carbohydrate may be used exclusively in the muscular 
process of breakdown and recovery, but as fast, or almost as 
fast, as it is used up it may be restored, in the general met- 
abolism of the body, by the breakdown of some other sub- 
stance, for example, of fat. The combustion of carbohydrate, 
followed by the reformation of carbohydrate from fat, would 
affect the respiratory quotient in a manner exactly similar 
to the direct combustion of fat. 


MECHANICAL EFFICIENCY ON DIFFERENT DIETS 


The beautiful and very convincing experiments of Krogh ~ 
and Lindhard, published in 1920, showed with little pos- 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 111 


sibility of doubt that the combustion of carbohydrate has 
some special connection with muscular activity. Employ- 
ing a method where the respiratory quotient may be deter- 
mined with an average error of only +0.002, measuring 
the cost of doing a given amount of work of moderate inten- 
sity in a highly trained and carefully observed and calibrated 
subject, and varying the substances metabolized by varying 
the diet during and before the experiment, they found the 
cost of work (that is, the total amount of energy used in 
doing a given amount of work) to be a linear function of the 
respiratory quotient, falling as the respiratory quotient 
rose. Of the total energy used in any effort, granting that 
the respiratory quotient be correctly measured, the fraction 
which is derived from fat may be shown to be a linear func- 
tion of the respiratory quotient. I say intentionally, 2f the 
respiratory quotient be correctly measured; we will discuss 
later the variations of respiratory quotient produced by 
lactic acid in the body during and after severe muscular 
work. Such variations, however, do not affect Krogh and 
Lindhard’s experiments, in which the exercise was moder- 
ate, requiring only about 1 liter of oxygen each minute, and 
continued for a long time. If now we assume that carbo- 
hydrate oxidized is utilized directly for work, or (more accu- 
rately) for recovery from work and the fat only after ‘‘con- 
version” involving metabolic processes and loss of energy, 
the cost of work should be a linear function of the respira- 
tory quotient—as Krogh and Lindhard found. As the mean 
of a long, careful, and carefully weighted series of observa- 
tions, which give one all the impression of extreme reliability, 


wee. 


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112 LECTURES ON NUTRITION 


they found, assuming carbohydrate to be utilized directly 
for the production of work (or, as I should rather say, for 
carrying on the recovery process) that fat may be so used 
only after “conversion” involving a 10 per cent loss of energy; 
in modern terms, the recovery process is 10 per cent less 


* efficient when fat is oxidized than when carbohydrate is 


oxidized. This suggests strongly that the primary break- 


_ down is of carbohydrate, and that fat is used only in a sec- 


ondary manner, for example, to restore the carbohydrate 
which has disappeared. These experiments of Krogh and 
Lindhard are particularly valuable since they were made on 
intact animals, namely, healthy men, and involved the com- 
plete process in the whole mechanism. 


LACTIC ACID 


The most important line of evidence in this connection 
starts from the work of Fletcher and of Fletcher and Hop- 
kins, leading to that of Meyerhof, Embden, and others. 
The phenomena of muscular fatigue are known to all, both 
personally and in the laboratory, as also is the effect of 
oxygen thereon. An isolated muscle stimulated in nitrogen 
soon fatigues and never recovers: an isolated muscle stim- 
ulated in oxygen may go on contracting for days. These 
observations of Fletcher led to the lactic acid story. In 
an isolated muscle at rest and without oxygen the acid ac- | 
cumulates slowly, faster at a higher temperature; with a 
sufficient supply of oxygen it remains at a low value. Stimu- 
lation also will produce lactic acid; in oxygen this lactic acid 
is removed. According to Embden and his co-workers, the 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 113 


origin of this lactic acid in the muscle is a hexose di-phos- 
phoric ester. They succeeded in isolating an osazone sim- 
ilar to that described by Harden and Young in the case of 
yeast. This hexose phosphate is presumably a very un- 
stable substance; it has not been isolated from muscle; its 
amount can be estimated only indirectly and on certain 
assumptions. Embden regards it as the immediate pre- 
cursor of the lactic acid which appears, though Meyerhof’s 
experiments make it clear that it is glycogen which bears a 
quantitative relation to lactic acid. The amount of Emb- 
den’s “‘lactacidogen” present in a muscle at any moment 
must be estimated by measuring the inorganic P.O; immedi- 
ately after, and one or two hours after, the fine division of 
the muscle: the increase in the P.O; is supposed to repre- 
sent the “lactacidogen” which has broken down. The evi- 
dence, though indirect, appears to yield results so definite 
that it is difficult not to believe that hexose phosphate is 
somehow intimately concerned with muscular activity. 
After severe muscular work, following a dose of phloridzin 
in rabbits, and after strychnine convulsions in rabbits and 
in dogs, there is a marked diminution in the “‘lactacidogen” 
present in their muscles. It is interesting too to record that, 
according to a communication of Robison and Kaye to the 
British Biochemical Society, the injection of insulin causes 
an increase in the “lactacidogen” of muscle. It must be 
admitted, however, that the réle of the hexose-phosphate is 
not yet clear. 


114 LECTURES ON NUTRITION 


THE CARBOHYDRATE ORIGIN OF LACTIC ACID 

It is very: natural to attribute a carbohydrate origin to 
the lactic acid which is concerned so intimately with mus- 
cular contraction. By the fermentation of various types of 
carbohydrate lactic acid may be formed, and Meyerhof has 
shown by a series of direct experiments, confirmed by inde- 
pendent methods at Cambridge by Foster and Moyle that 
, when lactic acid appears in muscle, whether from anaérobic 
conditions or from fatigue, an equivalent amount of glycogen 
_ disappears; in the converse process of recovery when the 
\ lactic acid is removed, glycogen reappears, not this time in 
equivalent amount, but with a 25 per cent loss, which is 
accounted for by the oxygen used and the heat produced in 
the recovery process. In the isolated muscle, therefore, | 
there can be no doubt that lactic acid has a carbohydrate 
origin and is restored to carbohydrate in recovery, a fraction 
of it only being used in the oxidative processes required to 
drive the recovery mechanism. That this recovery reversal 
of the glycogen-lactic acid breakdown is, at any rate in 
isolated muscle, carried out at the expense of energy derived 
from carbohydrate oxidation, is made the more certain by 
Meyerhof’s observation that the respiratory quotient of re- 
covery 1s unity. Moreover, in the isolated muscle there is 
no sign of any diminution in the fat contained in the mus- 
cle, as was shown by Winfield and confirmed to some degree 
by later and more severe experiments at Manchester (un- 
published). The total amount of glycogen present in a 
muscle is adequate to account for the whole of the energy — 
used in the most prolonged series of contractions that that 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 115 


muscle is capable of carrying out in oxygen, even under the 
most favorable conditions. It is possible, of course, that 
no transformation or combustion of fat is possible without 
the co-operation of other organs (for example, the liver) or 
of the body as a whole. We shall see later how far this ob- 
jection applies. In the isolated muscle, however, we may 
safely assert that the only processes which are known to 
occur, the formation of lactic acid from glycogen in the 
initial phase and the removal of the lactic acid, coupled 
with the oxidation of a small amount of it in the recovery 
phase, involve nothing but reactions with, and by, carbo- 
hydrate. 
PANCREATIC CONTROL 

Azuma and Hartree have shown that insulin has no effect 
whatever on the recovery oxidation in isolated muscle, and 
Foster and Woodrow that it has no effect on the lactic acid 
formation in resting surviving muscles. In intact animals 
under insulin treatment glycogen tends to disappear from 
the muscles (Dudley and Marrian), possibly partly to form 
a hexose phosphate, certainly not to form lactic acid. More- 
— over, Himwich, Loebel, and Barr have found that lactic acid 
formation in the diabetic individual is just as much the basis 
of muscular contraction as in the normal. This has been 
confirmed independently by my colleagues Long, Lupton, 
and Hetzel (hitherto unpublished), not only in the case of 
the formation of lactic acid, but in that of its removal in 
recovery. Apparently lactic acid is just as much involved in 
the mechanism of contraction in the diabetic as in the normal / 
man. That there is, however, some factor in the pancreas 


116 LECTURES ON NUTRITION 


concerned in the carbohydrate metabolism of muscle was 
shown by preliminary observations of Hopkins and Winfield 
in 1915, who found that pancreas preparations have an in- 
hibitory action on the formation of lactic acid in minced 
muscle. Apparently in the pancreas there is a substance, 
stable at high temperatures, which has a controlling action 
on the carbohydrate breakdown of muscle. This substance is 
not a ferment, and may be present in commercial pancreas 
preparations several years old. Foster and Woodrow fol- 
lowed up this clue and established the fact that there is an 
inhibitory agent for the anaérobic lactic acid formation in 
muscle which may be isolated from the pancreas and pro- 
duces considerable inhibition even under conditions leading 
usually to the maximal lactic acid formation. This sub- 


\ stance is not insulin, which has no such effect. They suggest 


that this new unknown substance has a specific controlling 
function on the carbohydrate metabolism of muscle, and 
that carbohydrate metabolism may be grouped into two great 
subdivisions, that of the body as a whole under the control 


of insulin, and’ithat of muscle, to some degree under the 


‘control of this new pancreatic hormone. 


ANALOGOUS METABOLISM IN OTHER CELLS 


An interesting side-line from Foster and Woodrow’s ex- 
periments arises when we remember that, according to the 
modern view of muscle, the basal metabolism of the intact 
animal is in large part the recovery from the resting lactic 
acid production of its muscles. This new pancreatic hor- 
mone might be expected, therefore, to control the basal 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 117 


metabolism, and possibly we may find in it a means of an- 
tagonizing an overactivity of the thyroid—though that is 
guesswork. [Foster and Woodrow, on the basis of these 
experiments, have put forward the theory of carbohydrate 
metabolism (not referred to) to which, on quite other grounds, 
those who have been working on muscular activity have been 
inevitably reduced, namely, that the carbohydrate metab- 
olism of muscle is a different thing from that of the body as 
a whole. That this carbohydrate metabolism of muscle, 
however, involving lactic acid is not unique is indicated by 
many lines of evidence. For example, Stephenson and 
Whetham have found that Bacillus coli, in a medium con- 
taining glucose, uses oxygen if it can get any and produces 
CO, and lactic acid; in nitrogen, COz2 and lactic acid are 
produced up to a certain limit, the fermentation being a 
self-inhibited one; in oxygen less lactic acid is produced, 
more CO, is liberated, and more oxygen is used. Apparently 
these organisms, in the presence of sufficient oxygen, can 
break down glucose completely to CO, and water. In the 
absence of sufficient oxygen they break it down, as does 
muscle, to lactic acid. If suspended in a medium containing 
no glucose but ammonium lactate, in the absence of oxygen 
they can do nothing; in the presence of oxygen they can 
produce CO, and use up oxygen, as does a muscle carrying 
out its recovery process. 

Again, Warburg, using the delicate gas-manometer method 
of Barcroft, has measured the COz produced and the oxygen 
used by various tissues suspended in a glucose-Ringer solu- 
tion. Some of the CO: is produced by combustion of carbo- 


118 LECTURES ON NUTRITION 


hydrate, some is driven out from preformed bicarbonate by 
acid formation. Expressing as “extra CO,” the amount of 
CO, produced in excess of that derived from the oxygen used 
in burning carbohydrate, the “extra CO,” is a measure of 
the lactic acid produced by the fermentation of glucose, 
that is, of the carbohydrate broken down, while the oxygen 
used is a measure of the carbohydrate oxidized. Normal tis- 
sues give a ratio, (extra CO2)/Os, of practically zero. Can- 
cerous tissues from a rat, however, give an. average ratio of 
3.6; human cancerous tissues give a ratio usually from 2 to 
4, but varying over wide limits. In normal tissues, there- 
fore, the oxidative process of glucose metabolism is effec- 
tive and the fermentative process is small; in cancerous 
tissues the oxidative process is ineffective and the fermenta- 
tive process is large. If Warburg is correct, the cancerous 
tissue zs like a muscle in which the recovery mechanism has 
almost broken down. 


GLYOXALASE 


In all tissues of the body except the pancreas Dakin and 
Dudley found a ferment, glyoxalase, which is capable of 
transforming methyl glyoxyl, CH;.CO.CHO, into lactic acid, 
CH;.CHOH.COOH. This ferment is inhibited by excessive 
acidity, that is, by an accumulation of the product of its 
own activity. It is present also in the blood of diabetic 
persons and in the blood and liver of diabetic dogs. An 
extract of the pancreas inhibits this reaction, and Dakin 
and Dudley have called this inhibiting substance antigly- 
oxalase. Antiglyoxalase is destroyed by heat, and on other 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 119 


grounds also it would appear not to be the same as the pan- 
creatic hormone of Hopkins and Winfield, and of Foster and 
Woodrow. It is conceivable that the normal path of carbo- 
hydrate metabolism may lie through the formation of lactic 
acid by glyoxalase from methyl glyoxal; unfortunately, ex- 
cept for the presence of this potent enzyme, glyoxalase, there 
is little evidence either for or against this theory. 


THE FATE OF LACTIC ACID IN RECOVERY 


The fate of lactic acid, which is an undoubted intermediary \ 
in the breakdown of carbohydrate in the muscle, was long : 
debated. .Its removal during recovery, as established by | 
Fletcher and Hopkins, was naturally credited at first to a 
simple process of oxidation. There were, however, certain 
fundamental difficulties about this to which there seemed 
to be no answer. The heat of combustion of glycogen, from 
which it is formed, is, according to Slater, 3,836 calories for 
each gram, when the glycogen is in its fully hydrated form, 
as it occurs in solution in the muscle; that of dissolved lactic 
acid is 3,601 calories (Meyerhof), leaving a total energy for 
the transformation of the one to the other of only 235 calories. 
This small quantity then is the total energy available in the 
initial transformation of glycogen to lactic acid, while in 
the complete process, if the lactic acid were then oxidized, 
3,836 calories would be liberated. The mechanical efh- 
ciency, therefore, of muscular contraction, supposing the 
whole of the initial energy were turned into work, could not 
exceed 6 per cent. Values of 25 per cent have been found 
in the case of man. Actually the initial liberation of heat? 


120 LECTURES ON NUTRITION 


for each gram of lactic acid formed in muscle Is larger than 
235 calories, being about 296 calories. ‘The difference has 
been attributed by Meyerhof to the neutralization of the 
acid by buffered alkaline protein salts inside the muscle 
fiber. There is no doubt that the acid is neutralized as soon 
as it is formed, since the hydrogen-ion concentration does 
not rise appreciably. Moreover, there is not enough phos- 
phate or bicarbonate present in the muscle to neutralize all 


“010 Rewative RATE oF RECOVERY [THE UNIT FoR EACH CURVE 15 
HEAT- PRODUCTION THE (INITIAL HEAT PER SECOND 


‘008 AT 20 Cc 


: SE 
O 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 


Fig. 19.—Delayed heat production of an isolated muscle in the presence of 
oxygen: C 0.03 sec. and D 0.20 sec. tetanus. (Hartree and Hill, 1922.) 


the lactic acid formed. Neutralization by phosphate and 
bicarbonate liberates only a little heat, while that by buffered 
protein salts liberates a large amount. Even assuming, 
however, that 296 calories are liberated in the initial phase 
and that the whole of this energy is turned into work, if the 
lactic acid were then oxidized the efficiency could still be 
only 8 per cent. Clearly the lactic acid is not oxidized. 

It has proved possible, moreover, to measure the total 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 121 


amount of heat liberated in the recovery process (Fig. 19), 
which in the latest and most careful measurements has come 
out almost exactly equal to the total heat liberated in the 
anaérobic phase. Thus, in the formation of and the sub- 
sequent removal of a gram of lactic acid only 740 calories of 
heat are found, which is only about one-fifth of what would 
occur if the lactic acid once formed from glycogen were 
subsequently oxidized im toto. Apparently, of every 5 mol- 
ecules of lactic acid removed in the recovery process only 
one is oxidized; the remaining four are restored to the place, 
or as the substance, from which .they arose. There is no pos- 
sibility of any error in the general conclusion to be drawn 
from these heat measurements: the difference to be explained 
is far too large; probably indeed the heat measurements 
provide us with the most accurate means of determining the 
“efficiency of recovery,” as we may call it (or, in Meyerhof’s 
term, the “oxidative quotient’’), that is, the ratio of the 
amount of lactic acid removed to that oxidized in its re- 
moval. The best value to assume seems to be, in frog’s 
muscle, about 5:1, and this is confirmed, as I shall show 
later, by two independent lines of experiment on man. This 
conception of the fate of lactic acid has been confirmed by 
Meyerhof’s direct observations of the glycogen restored and 
the lactic acid lost during the recovery process. All lines of 
experiment, therefore, on the isolated muscle indicate about 
the same value for the efficiency of recovery. There is no 
doubt that one must regard lactic acid in muscle as being 
not so much the fuel as part of the machinery. 


122 LECTURES ON NUTRITION 


THE RECOVERY PROCESS 

Consider now the recovery process in further detail. In 
the whole animal, without special precautions which we 
shall discuss later, it is not easy to isolate the recovery proc- 
ess from other events in the animal at large. In the isolated 
muscle the chemical method of investigation is not suffi- 
ciently analogous to what happens during normal existence, 
since the oxygen supply is cut off from its normal route by 
the cessation of the circulation, and (having to depend upon 
diffusion) is necessarily inadequate. The oxidative removal of 
lactic acid im 1solated muscles stimulated to severe fatigue has 
to take place under conditions of severe oxygen want, and 1s a 
very protracied affair. ‘The speeds, for example, at different 
temperatures cannot be compared, since they depend simply 
on the rate at which oxygen can pass in by diffusion from 
outside. Fortunately, another method is available which, 
compared with the chemical method, is of surpassing sen- 
sitivity, namely, that in which the heat production is meas- 
ured. Myothermic technic is so sensitive and so well under 
control that it is possible to measure and to analyze the 
course of the heat production for many minutes after only 
a single twitch of the muscle, in which case the total amount 
of energy involved and the total amount of oxygen used are 
so small that the amount of the latter originally dissolved in 
the fluid of the muscle is more than adequate to account for 
the whole of the oxidation carried out. We are independent, 
therefore, of the oxygen supply, and can study the speed and 
magnitude of the recovery process in a muscle provided with | 
an entirely adequate amount of oxygen. 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 123 


We find, when we stimulate a muscle, that there is initially 
a large production of heat, which must be attributed to the 
formation of lactic acid from glycogen and its subsequent 
neutralization. Then commences a slow process of recovery, 
in which heat is liberated continuously for many minutes 
until the muscle has been completely restored to its initial 
condition. The heat production rises rapidly at first, attains 
its maximum in a few seconds to half a minute, and then 
slowly falls to zero again, along a curve which is roughly 
exponential. This curve of recovery heat production is the 
thermodynamic outline of the recovery process, into which 
fuller details must be drawn later by biochemical analysis. 
The speed of the process depends on temperature: it is in- 
creased very largely by a rise of temperature, decreased by 
a fall, so that in a frog at 0° C. complete recovery, even from 
a few hops, must take an hour or more! Extrapolating the | 
results on frog’s muscle to the temperature of the human 
body, the recovery process from moderate effort should be 
nearly complete in two to three minutes, given an adequate 
supply of oxygen, as, indeed, we find it to be. Its speed 
depends also on the size of the initial breakdown of which 
it is the result, not only absolutely but relatively. Its speed 
is affected by the hydrogen-ion concentration, being dimin- 
ished by a rise and increased, up to a certain limit, by a 
fall, beyond which, however, it remains constant. Carbon 
dioxid, in concentrations of 10 to 15 per cent, produces a 
considerable fall in the rate of the recovery process, working 
much more quickly than do other acids, presumably because 
COz can more easily penetrate the muscle-fiber. The effect 


124 LECTURES ON NUTRITION 


of hydrogen-ion concentration on the speed of the recovery 
oxidation is analogous to that on the speed of autoxidation 
of glutathione or cystein. The total extra amount of heat 
liberated by oxidation in the recovery process is almost 
exactly equal to that set free in the anaérobic breakdown 
alone. 


THE “‘ACCUMULATOR FUNCTION” OF MUSCLE 


These facts have led us to the conception of the muscular 
machine as an accumulator of energy, analogous in its way 


.to a lead electrical accumulator. The initial discharge, 


hes wey 


which may take place at a high rate, depends in no way 


_ on the oxygen supply; the final recharge, which is slower, 
' depends directly on oxidation. In voluntary muscle all 


Saecese 


oxidation must be regarded as recovery oxidation: even 
though oxidation takes place during continuous exercise, 
and appears to be contemporary with the exercise, it must 
really be regarded as recovery from previous elements of the 
exercise. 

It is probably not true to assert that in all organs and 
tissues oxidation is recovery oxidation. For example, Starling 
and Verney have recently shown that in a kidney secreting 
normally the administration of KCN, which abolishes oxi- 
dation, produces immediately a change in the secretion, 
making it in all respects similar to a filtrate from the blood. 
Apparently ‘‘knocking out” oxidation immediately ‘knocks 
out”’ the capacity of the tubule cells to perform their normal 
function. It is probable that the same immediate depend- 
ence on oxidation exists in other tissues. Possibly those 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 125 


organs, in which sudden and violent activity may be re- 
quired at a moment’s notice and which are stimulated to 
activity through nerves, tend to act, as does voluntary 
muscle, like an accumulator; while slower tissues, in which 
rapid and violent response is not so necessary, may be con- 
tent to remain dependent for their energy on oxidation, as 
does an internal combustion engine. 


THE RECOVERY PROCESS IN MAN 


The conception that all muscular oxidation is really re- 
covery oxidation has produced an extensive change in out- - 
look in regard to respiratory experiments on man. One of 
the fundamental difficulties of a large animal is the supply 
of oxygen to his tissues. When muscular exercise starts the 
oxygen intake rises, attaining a maximum in man in two to 
three minutes. Respiration and circulation have to be 
worked up and the recovery process has to get under way. 
Muscles, however, are required for immediate and violent 
use, and even the maximal intake of oxygen, which in 
athletic men is about 4 liters per minute, can provide energy 
only for comparatively moderate exercise; in order to attain 
even that maximum a period of two to three minutes is 
necessary. Actually the human body is capable of exerting 
itself nearly ten times as violently as it could possibly do 
were it obliged to obtain all its energy immediately by com- 
bustion. Just as a lead storage cell is found to accumulate. 
sulphuric acid in the plates during its activity, so a muscle 
is found to accumulate lactic acid; just as the storage cell 
has its sulphuric acid removed from plates to solution dur- 


126 LECTURES ON NUTRITION 


ing recharging, so the muscle has its lactic acid restored to 
its precursor in recovery. With this conception it is of inter- 
est to study the process of recovery not only in isolated 
muscles, but in man, and in the last few years this study 
has proceeded a considerable way, especially by the efforts 
of my colleagues, Long and Lupton. Lupton, alas, has not 


+—S S Breathing 497 Oxyger 


Broken lines | Show Period of | Exercise 


7ime trem commencement of Excercise Minutes 


10 20 30 40 50 60 


Fig. 20.—Lactic acid in human blood after severe muscular exercise; 
two experiments in air, one in 49 per cent oxygen, one in 100 per cent 
oxygen. Note that the recovery process is not quite complete at the end 
of the time shown in the diagram. (Hill, Long, and Lupton, 1924.) 


lived to reap the reward of his devoted work, or to realize 
the full importance of what he did. 


LACTIC ACID IN MAN 


Lactic acid may be studied directly in man by its estima- 
tion in blood removed in the usual way from a vein. During 
muscular activity the lactic acid in the blood rises, attaining 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 127 


finally, if the exercise be continued long enough, a maximum 
characteristic of the effort made. After a while the lactic 
acid distributes itself by diffusion equally in all tissues which 
are directly in contact with the blood-stream. During re- 
covery this lactic acid disappears, in a period depending on 
the severity and duration of the preceding exercise, but not 
exceeding in normal man about ninety minutes (Fig. 20). 
The removal of lactic acid from the blood, which is a sign of 
its preceding removal from the muscle, is produced by oxi- 
dative processes occurring in the latter. These oxidations 
can be studied by ordinary respiratory methods, employing 
the Douglas bag technic. The initial phase of the recovery 
process, which is-rapid and is concerned with the oxidative 
removal of the acid in the muscles where it was formed, can 
be followed by means of collections in a series of bags. The 
recovery oxidation falls rapidly, and after moderate exer- 
cise reaches zero in a few minutes. If, however, the exercise 
was severe, the lactic acid will have had time to escape from 
the muscles into the blood, and into other tissues in con- 
tact with the blood, and a second phase of recovery will 
occur, the removal of lactic acid which has escaped. This 
second phase may be very protracted and last as long as 
eighty minutes. The total oxygen used in the recovery 
process in this way we have named the “oxygen debt at the 
end of exercise.’ Assuming, what may be shown to be very 
nearly true, that it is all used in the oxidative removal of 
lactic acid, and employing a value of 5 : 2 : 1 for the efficiency 
of recovery, we may calculate from the oxygen debt the lac- 
tic acid present in the body at the end of exercise. We find 


128 LECTURES ON NUTRITION 


that 3 gm. or more of lactic acid may be liberated each second 
in the muscles of a powerful man, and that the body is able 
to tolerate an amount up to a Zotal of 130 gm. The oxygen 
debt may attain a value of 18.7 liters! 

This lactic acid formation, therefore, in the human body 
is not a small or unimportant factor in muscular exercise; 
it is the keystone of the whole structure and has a large, 
indeed, a preponderant, effect on the respiratory quotient.” 


EXP! Very Violent Exceruse for 36 secs ae 
aN 
E> 


Broken Lines show Period of Excerchs 


EXP 2. Moderate Excercise for 4% mins. 


2 4 6 8 Te) 12 4 16 18 20 


Fig. 21.—The respiratory quotient during and after muscular exercise. 
These figures show the initial phase of recovery only. ‘The final phase is 
shown in Fig. 22. (Hill, Long, and Lupton, 1924.) 


° 
G 


Respiratory Quotient | = 
(0;/0, 


The respiratory quotient varies in a striking manner, up and 
down, during the onset of severe exercise and in recovery 
from it. At first it rises (Fig. 21), attaining a value up to 2, 
during and immediately after the phase of lactic acid lib- 
eration, and while the respiratory center is still endeavoring 
to cope with the increased hydrogen-ion concentration of 
the tissues. Before the hydrogen-ion concentration of the 
body can have returned to its previous resting value an 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 129 


amount of CO. must be driven off equivalent to the lactic 
acid still present. After this previous level of the hydrogen- 
ion concentration has been attained, which happens several 
minutes after recovery has commenced, the lactic acid con- 
tinues to decrease and CO, has to be retained by the body, 
since otherwise the latter would become far more alkaline 
than previously. In the later stages of recovery the CO2 


08 


Respiratory Quotient 


0-6 


Fig. 22.—The respiratory quotient after severe eh exercise. Note 
that in the later phases, while carbon dioxide is being retained to com- 
pensate for that initially driven off, the respiratory quotient falls to a 
very low level, returning to its final value at about eighty minutes. (Hill, 
Long, and Lupton, 1924.) 


retained is a measure of the lactic acid removed and very low 
values of the respiratory quotient may be found, down to 
0.6 (Fig. 22). Assuming that CO, retention to be a measure 
of the lactic acid removed, and the oxidation in excess of 
the basal value a measure of the lactic acid oxidized, we 
may determine in man the efficiency of recovery by respira- 
tory methods, and its value comes to about 5:1, the same 


as in isolated muscle. Another method may be used in 
9 


130 LECTURES ON NUTRITION 


estimating the same quantity in man. If the lactic acid 
found in blood be assumed, in the later stages of recovery, 
to be uniformly distributed in all the soft tissues of the 
body which are in immediate contact with the blood-stream, 
we may calculate by two observations of the blood over 
any interval the total amount of lactic acid removed, and 
from the excess oxygen used in that interval we may again 
determine the ratio of lactic acid removed to lactic acid oxi- 
| dized. We find as before a value of about 5:1, so that in 
the complete and intact animal the mechanism of recovery 


é£ 


‘appears to be the same as in the simple isolated muscle. 


THE USE OF THE RESPIRATORY QUOTIENT 


These large variations in the respiratory quotient, during 
severe exercise and in recovery therefrom, show how neces- 
sary it is to exercise the greatest possible precautions if we 
wish to draw any conclusions from the respiratory quotient 
as to the substance being oxidized. Such precautions were 
taken in the experiments of Krogh and Lindhard. The exer- 
cise must be moderate and very long continued, and the whole 
condition of the subject must be “‘steady.”’ Then only are 
deductions reliable; otherwise the value of the respiratory 
quotient tells us more about the fluctuations of lactic acid in 
the body than about the nature of the metabolism. 


THE OXYGEN “REQUIREMENT” OF EXERCISE 


When muscular exercise commences the oxygen intake 
rises to a value which is either the equivalent of the exer- 


cise, if the latter be moderate, or is the maximum character: 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 131 


istic of the individual subject, if the exercise be severe. In 
the latter case the exercise can be continued only for a time, 
the lactic acid accumulates, fatigue comes on, and the mus- 
cles finally are incapable of further effort. The oxygen intake 
is a measure of the severity of the exercise only if the latter 
is sufficiently protracted to enable a steady state to be at- 
tained, and sufficiently gentle to ensure that there is not a 
constant accumulation of acid leading to an oxygen debt. 
Hence by a study of the oxygen intake and the CO, output 
we can never really determine the nature of the primary 
oxidations of muscular activity, since the exertion must be 
continued for a long time until the body and all its processes 
are in a steady state, and the primary reactions of muscular 
recovery may then be masked by other and secondary effects. 
The oxidation of carbohydrate required to drive the recovery 
process may be confused, for example, with the re-formation 
of carbohydrate from fat. 

This fact and others have led us to a study of what we 
call the “oxygen requirement.” The subject of the experi- 
ment takes exercise of any character and of any duration, 
the total oxygen used during the exercise and in complete 
recovery from it being measured. An initial and a final 
estimate of the resting oxygen consumption give us a base 
line from which the total oxygen consumption resulting from 
the exercise, during and in recovery from it, may be cal- 
culated. The measurement of the oxygen requirement is 
valuable, since it can be made in the case of any type of 
exercise, for example, walking up a single flight of stairs, 
or in very violent exercise which could not be continued long 


132 LECTURES ON NUTRITION 


enough to make a measurement of the oxygen intake pos- 
sible or useful. It may be a valuable criterion of the mechan- 
ical efficiency of work, etc.,; The oxygen requirement for a 
short element of exercise is always a measure of the total 
amount of energy required by the body for that exercise, 
assuming, as we shall see below, the energy value for oxygen 
corresponding to the oxidation of carbohydrate. 


THE RESPIRATORY QUOTIENT OF EXERCISE AND RECOVERY 


Much greater interest attaches to the respiratory quo- 
tient when we consider not only exercise, but subsequent 
recovery. ‘Taking the case of a small element of muscular 
exercise, such as running slowly for thirty seconds, the rest- 
ing respiratory exchanges are measured carefully, both be- 
fore the exercise and after complete recovery. The expired 
gases are collected both throughout the exercise, and during 
a recovery interval sufficiently long to ensure that the me- 
tabolism has returned absolutely to its initial state. The 
excess oxygen used as a result of the exercise and the excess 
COz given out are then determined by analysis and calcula- 
tion: they are found to be precisely equal. The same is true 
of fairly violent exercise for a short interval. In the case, 
however, of very violent exercise, the recovery process may 
be very protracted and the respiratory quotient of the ex- 
cess metabolism may be less than unity. If the oxygen 
requirement of a very long period of exercise be measured, 
it is obvious that the respiratory quotient will not be unity. 
In such a case nearly all the excess of oxygen used and of 
CO, produced by the exercise occurs during the latter, while 


MUSCULAR ACTIVITY, CARBOHYDRATE METABOLISM 133 


the respiratory quotient is, say, 0.85. Thus, as we should 
expect, when a bout of exercise is increased in duration from 
very short to very long, the respiratory quotient of the 
complete cycle passes gradually, from a value of unity for 
the very short, to a lower value characteristic, as in the 
experiments of Krogh and Lindhard, of prolonged steady 
exercise. These results appeared first incidentally in a 
study of the oxygen requirement of exercise, carried out for 
another purpose. We noted, however, that of about twenty 
experiments practically all gave a respiratory quotient of 
the excess metabolism of about unity, the mean value being 
1.03. The small excess we attributed to the fact that in 
these experiments recovery was not quite complete, and the 
small CO, retention of the last phase had not come within 
our observation. Since then my colleague, Dr. Furusawa, 
has examined the matter more carefully. His experiments 
(Tables 1 and 2) show that the respiratory quotient of the 
excess metabolism due to a short element of muscular exer- 
cise is unity. This is the case even if the subject, having 
lived for several days on a diet of fat and protein, has a 
resting respiratory quotient of little more than 0.71 (Table 
2). If the exercise be prolonged the stores of carbohydrate 
are used up and have to be reformed by the transformation 
of other substances, presumably of fat; such a transforma- 
tion acts on the respiratory quotient just as though fat 
itself were being oxidized, so that the respiratory quotient 
falls. Given, however, an element of muscular exercise, so 
moderate in duration and severity as to produce no meas- 
urable carbohydrate lack in the muscles and no disturbance 


134 LECTURES ON NUTRITION 


TABLE 1 
(Unpublished experiments by K. Furusawa) 
NorMAL DIET 


R. Q. of Excess Metabolism Due to Exercise 


Duration Steps Duration Excess 
of per of collec- metabolism, 
exercise, minute. tion, minutes. COz /Oz. is (Op 
minutes. 
0.5 92 10 355/350 1.00 
0.6 64 10 485/490 0.99 
1.0 146 20 2120/2037 1.04 
RA US 160 31 4185/4048 1.03 
2.0 208 76 11230/10251 1.09 
10 120 60 8720/8900 0.98 
LZ 160 85 - 19860/18620 1.06 
13 146 hit 25670/25960 0.99 
20 146 110 41310/42210 0.98 
28 146 63 49520/52900 0.94 
30 120 70 20225/20345 0.99 
30 146 105 49420/56230 0.88 


Not postabsorptive, but several hours after a meal. 
Average R. Q. resting, 0.85 
Average R. Q. excess metabolism = 1.02 
(Up to 30 liters Qy.) 


of metabolism in the rest of the body, we find that the whole 
oxidative cycle of recovery is carried out at the expense of car- 
bohydrate. 

This confirms and amplifies the experiments of Krogh 
and Lindhard with which we started this discussion, and 
makes clear how the experiments on the isolated muscle, 
with their indication that carbohydrate is the essential fuel 
of muscle, may be reconciled with the respiratory quotients 
obtaining in prolonged moderate exercise in man. T he 
primary fuel of muscle 1s carbohydrate; the essential element 
in the machinery 1s lactic acid, itself derived from carbohy- 


MUSCULAR ACTIVITY, CARBOHY DRATE METABOLISM 135 


TABLE 2 
(Unpublished experiments by K. Furusawa) 
Fat DIET 
(Bacon, Butter, Milk, Fat Chops) 
R. Q. of Excess Metabolism Due to Exercise 


Duration of Steps Duration of Excess Compare 
exercise, per collection, metabolism, RAO! 

Subject. minutes. minute. minutes. CO2 /Oz. REOs at rest. 
Ie 0.33 272 22 2733/2570 1.06 0.72 
K. F. 1.0 146 20 2400/2260 1.06 0.71 
K. F. 2.0 146 30 3020/2964 1.02 0.75 
Ke. 4.0 146 43 10525/10822 0.97 0.78 
Ae) Oe 7.0 146 50 13730/15145 0.91 0.72 
Kok 9.0 146 100 18910/19940 0.95 0.76 
se bs 9.0 146 88 18835/20420 0.92 0.77 
Jai? P: 0.4 216 22 3260/3341 0.98 27h 
FSUCP EE LO.5 196 25 2132/1966 4.087107 
A Ss ed 1.0 146 26 3300/3345 0.99 0.73 
LEP. 0 162 24 5255/5256 1.00 0.75 
you Powesed 146 8 273500 1.2910 113775) 099401. 0.75 


Fatty diet, K. F. five days, J. L. P. three days, before experiments. 


drate. ‘The breakdown of carbohydrate in muscle is asso- 
ciated with the presence of phosphates, possibly in the form 
of a hexose diphosphoric ester. What the further details 
of the process are we do not know, but it is difficult not to 
believe that the utilization of fat by muscles can occur only 
after its previous ‘‘conversion” somewhere in the body. 
Even a subject suffering from severe carbohydrate want 
(Table 2) will oxidize carbohydrate, and carbohydrate alone, 
in the complete cycle of reactions resulting from an “ele- 
ment’’ of muscular exercise. 


BIBLIOGRAPHY 
1. Azuma and Hartree: Biochem. Jour., 17, p. 875, 1923. 
2. Dakin and Dudley: Jour. Biol. Chem., 14, pp. 155 and 423, 1913; 15, 
p. 463, 1913. 


136 LECTURES ON NUTRITION 


3. Dudley and Marrian: Biochem. Jour., 17, p. 435, 1923. 

4, Embden and Laquer: Zeitsch. physiol. Chem., 113, pp. 1-9, 1921. 
5. Embden, Schmitz, and Meincke: Ibid., 113, pp. 10-66, 1921. 

6. Fletcher: Jour. Physiol., 28, p. 474, 1902. 

7. Fletcher and Hopkins: Ibid., 35, p. 247, 1907. 

8. Foster and Moyle: Biochem. Jour., 15, p. 672, 1921. 

9. Foster and Woodrow: Ibid., 18, p. 562, 1924. 

10. Hartree and Hill: Jour. Physiol., 56, p. 367, 1922; 58, p. 127, 1923. 

11. Hill: Jour. Physiol., 48, p. xi, 1914. 

12. Hill, Long, and Lupton: Proc. Roy. Soc., 96B, p. 438, 1924; and two 
other instalments in the press. 

13. Hill and Lupton: Quart. Jour. Med., 16, p. 135, 1923. 

14. Himwich, Loebel, and Barr: Jour. Biol. Chem., 59, p. 265, 1924, 

15. Hopkins and Winfield: Jour. Physiol., 50, p. v, 1915. 

16. Krogh and Lindhard: Biochem. Jour., 14, p. 290, 1920. 

17. Meyerhof: Pfliiger’s Arch., 175, p. 88, 1919; 182, p. 232, 1920; Ibid., 
p. 284; 185, p. 11, 1920; 195, p. 22, 1922; 204, p. 295, 1924; Biochem. 
Zeitsch., 129, p. 594, 1922. 

18. Slater: Biochem. Jour., 18, p. 621, 1924. 

19. Stephenson and Whetham: Biochem. Jour., 18, p. 498, 1924. 

20. Warburg: Klin. Wochensch., No. 24, 1924. 

21. Winfield: Jour. Physiol., 49, p. 171, 1915. 


OUR PRESENT KNOWLEDGE OF THE VITAMINS* 


ELMER VERNER McCoLitum 


In presenting the position of our knowledge of the vita- 
mins, one is required to tell what we know of a class of sub- 
stances of unknown nature which are widely distributed in 
our foodstuffs, and whose physiologic function we do not 
understand. We do not know whether the vitamins are 
essential, in certain cases at least, for the general biologic 
processes of cell activity, or whether they exert their action 
on cells of specific types. The methods of study of the dis- 
tribution, chemical nature, and mode of action of the vitamins 
parallel in many respects those applicable to the study of 
the hormones. Indeed, the phenomena relating to the vita- 
mins are so suggestive of the hormones that the term “food 
hormones” has been used by some as synonymous with vita- 
mins. In either field of inquiry we recognize the existence 
of a substance which is indispensable for normal metabolism 
by observing what happens when it is not present. With 
certain of the hormones it is possible to study the effects of 
an excessive amount of a product elaborated by a particular 
tissue. In the field of vitamin study no one has demonstrated 
as yet any effects of administering large amounts of the 
active substances. 


* A more extended discussion will be found in the third edition of The 
Newer Knowledge of Nutrition. 
137 


138 LECTURES ON NUTRITION 


It is now twenty-six years since it was clearly demonstrated 
by Eijkman, Grijns, Hulshoff-Pol and Schaumann, working 
in Batavia and Java, that beri-beri was caused by lack of 
something in the diet which could not be supplied by polished 
rice, but which was present in relative abundance in the 
polishings of rice and in “katjang hidjoe” bean. Ten years 
later Funk (1913-1915) repeated and confirmed the obser- 
vations of the Dutch investigators. He suggested the name 
““vitamine”’ to designate a class of substances which he believed 
to be necessary for the prevention of beri-beri, scurvy, rickets, 
pellagra, and sprue, respectively, as well as of another or others 
necessary for growth as distinguished from maintenance. 
Funk’s investigations created a new interest in studies of 
nutrition. At that time it was only possible to demonstrate 
either a preventive or curative action of a vitamin prepa- 
ration in the case of beri-beri, or polyneuritis, as it is called 
when produced experimentally in an animal. The substance 
which is active in the restoration of a pigeon suffering from 
polyneuritis is now known as vitamin B. From this begin- 
ning progress has been steady, until today, almost all inves- 
tigators believe the evidence is conclusive that for one or 
another species of animal five vitamins are necessary. Be- 
sides these principles, which have been designated as vita- 
mins A, B, C, D, and E, another substance called bios is 
believed to exist, which has been confused with vitamin B. 


VITAMIN A 


David Livingstone, in describing the hardships endured in 
his explorations in Africa, called attention as far back as 


OUR PRESENT KNOWLEDGE OF VITAMINS 139 


1857 to a peculiar eye condition from which his party suffered 
while restricted to a diet of sugarless coffee, manioc roots, and 
meal, and said, ‘‘the eyes become affected as in the case of 
animals fed in experiment on pure gluten or starch.” This 
appears to be the earliest mention of the ophthalmia which 
is the most characteristic pathologic change which results 
from a lack of the vitamin A in the diet. I have been unable 
to discover a description of the experiments to which Living- 
stone refers. Although ophthalmologists have associated for 
many years keratomalacia with faulty diet, as, for example, 
among the Russian peasants during religious fasts, the first 
definitely to associate xerophthalmia in the human subject 
with faulty diet was Mori (1904). He described about 1,500 
cases of xerophthalmia or keratomalacia among children in 
Japan between the ages of two and five years from 1905 to 
1907 inclusive. He pointed out that the eye symptom was 
readily relieved when cod-liver oil was administered, and 
called attention to the popular belief in the efficacy of chicken 
livers and eel fat as a remedy for this eye disease in children. 

Falta and Noeggerath (1906), before the vitamin hypothesis 
was formulated, described a conjunctivitis with crust forma- 
tion beginning about the fourth week and lasting until death 
in rats which were fed a diet which from their description 
must have been very deficient in vitamin A. Several other 
observers subsequently called attention to an edema of the 
eyelids and xerosis of the conjunctiva in animals fed a diet 
which we now know was lacking in vitamin A. 

Stepp (1909) was the first to show by toneligiee experi- 
ments that some nutrient principle not identifiable with any 


140 LECTURES ON NUTRITION 


of the known lipins was necessary for the maintenance of 
health in grown mice. Stepp’s work was supplemented in 
1912 by the author and by Fingerling (1912), who demon- 
strated independently that all the complex lipins which occur 
in the egg yolk or which are necessary for growth in birds can 
be synthesized from some non-lipin substances in the diet. 
Up to 1912 all students of foods believed that fats had es- 
sentially the same nutrient qualities irrespective of their origin 
or nature. The demonstration in 1913 that certain fats had 
nutritive qualities not possessed by other fats equally digest- 
ible and palatable marked a milestone in the progress of nu- 
trition studies, since it showed that by properly planned ex- 
periments on animals hitherto unsuspected qualities in food- 
stuffs could be demonstrated. McCollum and Davis (1913) 
were the first to plan an experiment which conclusively showed 
that butter fat and the fats of egg yolk contain something 
which is essential for the nutrition of the rat and which could 
not be provided by including lard or olive oil in the ration. 
Soon afterward Osborne and Mendel confirmed these obser- 
vations on butter fat and lard, and added almond oil to the 
list of fats which were ineffective in inducing growth. They 
showed that cod-liver oil possesses the same properties as 
butter fat and egg yolk fat, and called attention to what they 
believed to be an infectious eye disease which was relieved 
by the administration of the “growth-promoting fats.” 
Bloch observed many cases of xerophthalmia in children 
in the vicinity of Copenhagen during the years 1912 and 1916. | 
These children were fed a diet consisting of separator skimmed 
milk practically free from fat. The milk had been pasteurized 


OUR PRESENT KNOWLEDGE OF VITAMINS 141 


and cooked again in the home. Oatmeal and barley soup 
constituted the other important ingredients of the diet. 
Bloch referred the malnutrition in these infants to lack of 
fat in the diet, and observed that the eye trouble could be 
relieved by the administration of cod-liver oil, whole milk, 
or cream mixtures. 

During a period of several years McCollum and Simmonds, 
who had repeatedly observed this peculiar form of ophthalmia 
in animals subjected to faulty diets, collected numerous ob- 
servations on the experimental groups of rats in their colony 
where the ophthalmia occurred. A study of the data revealed 
the fact that many groups of rats in various degrees of en- 
feeblement or arrested development, the result of restriction 
to faulty diets of various kinds, failed to develop the disease, 
while others in adjacent cages were suffering from it. They 
found the ophthalmia to occur only in animals whose diets 
were deficient in the vitamin A, and in 1917 they correlated 
their observations on rats with those reported by Bloch 
and Mori, and expressed the view that the xerophthalmia 
or keratomalacia produced experimentally in animals is the 
analogue of the condition which these authors had reported 
in man. Since the publication of these observations the 
development of xerophthalmia has been generally accepted 
as the most characteristic sign of a lack of the vitamin A. 

The view has not infrequently been expressed by clinicians 
that the ordinary mixed diet on which we live is satisfactory 
for the promotion of health, since the well-marked deficiency 
diseases occur only among people whose diets are notably 
restricted and monotonous. Evidence has been steadily 


142 LECTURES ON NUTRITION 


accumulating that this view is fallacious. As an illustration, 
Cramer (1924) describes epidemics of eye affections which 
occurred in England during the last forty years in various 
industrial schools. The last recorded outbreak was in 1911 
and was investigated by McNeil and McGowan (1913). 
The condition of the children was diagnosed as “distorted 
pneumonia” which varied from a fulminating rapidly fatal 
type to an abortive or latent type. Special mention was 
made of the prevalence of a chronic granular conjunctivitis. 
Investigators clearly recognized the non-contagious nature 
of the epidemic, but were unable to explain the nature of the 
disease or to recommend a rational treatment. From our 
present knowledge of quality in foods it seems highly probable 
that these boys were manifesting the effects of vitamin A 
starvation. Several observers have correlated faulty dietary 
with the incidence of hemeralopia. Such relationship was 
described in 1883 by DeGouvea, who observed the disease 
among the negro slaves working on coffee plantations of 
San Paulo, Brazil. Little, Grenfell, Appleton, and others 
have mentioned the prevalence of night blindness in New- 
foundland and Labrador. Blegvad (1923) studied the utili- 
zation of a preparation of vitamin A when injected subcuta- 
neously. He estimated his preparation to have about 100 
times the potency of cod-liver oil. He asserts that the util- 
ization is much more rapid when this vitamin is introduced 
subcutaneously than when fed by mouth. 

Osborne and Mendel (1917) observed calculi of calcium 
phosphate in the urinary tract in ninety-one animals among 
857 necropsies. Forty-three per cent of these had not had a 


OUR PRESENT KNOWLEDGE OF VITAMINS 143 


satisfactory supply of vitamin A. In McCollum and Sim- 
monds’ experience calculi have occurred so frequently in 
animals whose diets contained an abundance of this factor, 
but were faulty in other respects, that it would seem to be 
the result of general debility rather than lowered vitality 
brought about by specific cause. 

Wason (1921) studied the pathology of ophthalmia of diet- 
ary origin. She observed in the eyes of rats in which the dis- 
ease was induced by selective fasting for vitamin A, hyalin- 
ization or necrosis of the outer layer of corneal epithelium, 
exudation of serum and tells into epithelium and stroma, and 
a proliferation of blood-vessels and fibroblasts. In advanced 
cases invariably the anterior and occasionally the posterior 
chambers were invaded. She concluded that the type and 
virulence of the organisms of secondary infection determine, 
in part at least, the course of the disease. 

Yudkin and Lambert (1922) found that the changes in 
the eyes resulting from lack of vitamin A do not begin in 
the cornea, but have their origin in the lid. In this respect the 
sequence of events is the same as that of some of the severer 
types of acute and chronic conjunctivitis which are frequently 
complicated by corneal injury with infection and ulceration 
of this structure. They also say that the lacrimal gland may 
be the seat of marked pathologic changes either degenerative 
or inflammatory in nature. They observed variation in 
size and form and in staining properties of the cells, which 
they refer to functional disturbances related to the ophthalmia. 

Mori (1922, 1923) studied histologically the eyes of rats 
in various stages of specific starvation for vitamin A. He 


144 LECTURES ON NUTRITION 


found the first observable change to be a tendency for the 
lacrimal gland to go into a resting stage, and to cease to 
produce tears. He believes that subsequent changes in the 
eyes can be accounted for as the result of loss of function 
of the tear glands. When the supply of tears fails the con- 
junctival sac is no longer washed continuously as under 
normal conditions, and bacteria quickly begin to grow there 
in great numbers. The growth of bacteria stimulates a 
migration of leukocytes, which accumulate in the eye chamber, 
causing the well-known hypopyon, which is visible as a yel- 
lowness in the pupil. Some of the leukocytes migrate through 
the outer coating of the eyeball and find their way into the 
conjunctival sac. Through their dissolution albuminous ma- 
terial accumulates which forms a sticky exudate that tends 
to paste the eyelids together. The drying of this exudate 
makes it difficult for the animals to get their eyelids open. 
It was found possible to cultivate ever-increasing numbers 
of bacteria from swabs taken from the surface of the eyeball 
day by day during the progressive fasting for vitamin A. 
Owing to dryness of the eye there is cornification of the ex- 
ternal coating, the cells become flattened and pile up in 
pseudo-stratified form, resembling horny layers of skin. 
Ulcers regularly form on the cornea during the later stages 
of the disease owing to the death of the tissue. The ulcers 
finally perforate and the lens pops out. It is possible to in- 
duce spectacular recovery in animals whose eyes are in a 
very damaged condition by administering fats containing an 
abundance of vitamin A. Mori regards the ophthalmia of 
dietary origin as an analogue in certain respects at least of 


OUR PRESENT KNOWLEDGE OF VITAMINS 145 


a human disease common in many parts of the Orient and 
known as hikan. 

Sections of the lids of the eyes of animals suffering from 
xerophthalmia very often show cystic dilatation of the ducts 
of the meibomian glands. These cysts are filled with fat, 
the secretion of the gland cells. The epithelium of the margin 
of the lid shows evidence of a very marked xerotic process, 
and the same change is found in the epithelium of the duct. 
It is very likely that this xerosis may play a part in the 
occlusion of the duct and the formation of the retention 
cysts. 

The lumina of the acini of the harderian gland may be 
either very much dilated and empty, or narrowed by swelling 
of the secreting cells. Frozen sections of this gland show a 
remarkable diminution of the fat content of the cells and the 
lumen. The connective tissue about the gland acini is very 
often densely infiltrated with round cells. 

The mucous cells of the conjunctiva are all entirely des- 
troyed in the course of the xerotic process. It is certain from 
these findings that the entire secretory apparatus of the eyes 
of these rats is in a state of dysfunction. The secretion from 
these glands is either very much diminished or entirely lack- 
ing. The changes in the lacrimal gland are the most im- 
portant in the pathologic picture of this disease, and, in fact, 
the changes in this gland would seem to be the cause of the 
lesions in the cornea and conjunctiva. The diminution or 
the lack of the secretion of the lacrimal gland would explain 
the dryness of the eyeball as well as the xerosis of the cornea 


and the conjunctiva. The failure of the lacrimal secretion 
10 


146 LECTURES ON NUTRITION 


also explains the increase in number of the organisms found 
in the conjunctival sac. 

Mori also examined the salivary glands of rats suffering 
from lack of vitamin A, since both in nerve supply and in 
structure these resemble closely the lacrimal gland. The 
submaxillary and parotid glands of the rat are serous; the 
sublingual is a mucous gland. In many rats with xeroph- 
thalmia either all or some of these glands are either not se- 
creting at all or secreting very little. The secreting cells 
become much shrunken, the acini are very small and show no 
traces of secretion. The epithelium of the intralobular ducts 
is shrunken and the cells are irregular in size. 

The epithelium of the principal ducts of these glands often 
shows cornification and desquamation of the thickened su- 
perficial cells, so that the lumen of the duct becomes narrow 
and is often occluded. Dilatation of the small ducts occurs 
frequently in the parotid gland. Coincident with the xerotic 
changes and those of the parenchymal cells, the ducts are 
invaded by bacteria, and small abscesses are formed in the 
gland. 

The other secretory organs, such as the liver, pancreas, 
bowel, and kidneys, showed no remarkable change except in 
one case. In one antmal the cytoplasm of a small number of 
cells of the pancreas was very much vacuolated. ‘The re- 
productive glands of the rat on a diet deficient in vitamin A 
do not function. 

Evans and Bishop (1922) found a characteristic disturbance 
of the cestrual cycle in the rat caused by a deficiency of vita- 
min A. ‘This resembled no other nutritional upset known. 


OUR PRESENT KNOWLEDGE OF VITAMINS 147 


It consisted of a prolongation of the cestrual desquamative 
change in the vaginal epithelium. The smear consisted 
chiefly, if not exclusively, of cornified cells which in the 
normal individual characterize the actual period of cestrus 
and ovulation only. In the case of animals showing a de- 
ficiency of vitamin A these cells occur during the entire 
period of the acute deficiency. This, they state, may con- 
stitute the only sign of deficiency of vitamin A except the 
failure to reproduce successfully. 

Cramer (1923) studied the pathologic lesions induced by 
deficiency of vitamin A and interprets his observations as 
pointing to the digestive tract as the key to the problem of 
the mode of action of this accessory substance. He believes 
the functional integrity of the digestive tract to be dependent 
on the presence in the food of certain vitamins. He believes 
these have a specific drug-like action analogous to the effect 
on the functional inactivity of the uterus by a hormone pro- 
duced in the ovary. The vitamin A has, according to his 
view, a specific stimulating effect on the intestinal mucous 
membrane, and also directly or indirectly on the formation 
of blood platelets. Cramer’s histologic studies on sections 
of the rat’s intestine, made “upward from the cecum,” indi- 
cate that on a vitamin-free diet, about the time xerophthal- 
mia begins to develop, there is seen a profound atrophy of 
the villi and necrosis of the upper parts of them. Rats in 
an advanced stage of deficiency of vitamin B, but receiving 
an abundance of vitamin A in cod-liver oil, had villi which de- 
viated in appearance from the normal, but showed no atrophy 
and no necrosis. Cramer asserts that the vitamins have a 


148 LECTURES ON NUTRITION 


positive action, and he believes he has demonstrated an 
important stimulating effect on the process of food absorp- 
tion from the intestine in perfectly normal animals, which 
have never been subjected to any dietetic deficiency, when 
the vitamin moiety in their diet is markedly increased. 

Besides these gross changes an abundance of protozoa, 
mainly Gzardia intestinalis, were found in the lumen of the 
intestine of rats in advanced deficiency of vitamin A. These 
are presumably present in small numbers in the intestine, 
but the absence of vitamin A enables them to proliferate 
rapidly and to penetrate between the villi where normally 
they are never present. This fact, Cramer points out, may 
be of importance in determining the pathogenicity of certain 
organisms. They may be non-pathogenic on one diet and 
become pathogenic by variation of the pabulum in the intes- 
tine. 

The intestinal bacteria also appear to increase greatly in 
number over what is normally seen, and, what is more signifi- 
cant, may often be seen adherent to the villi, especially the 
necrotic tips. Normally the bacteria remain confined to the 
center of the lumen. In deficiency of vitamin A Cramer 
found the bacteria often invaded the mucous glands and were 
found in dense masses filling the lumen of the gland to the 
bottom. 

Mention has already been made of the view of Cramer 
that, among other effects, the vitamin A produces directly 
or indirectly a stimulation of the formation of blood plate- 
lets. Cramer, Drew, and Mottram (1922) reported that a 
diet deficient in vitamin A produces a progressive decrease 


OUR PRESENT KNOWLEDGE OF VITAMINS 149 


in the number of blood platelets, and that thrombopenia 
was the only constant lesion for vitamin A deficiency and 
was characteristic of this deficiency just as lymphopenia was 
characteristic of vitamin B deficiency. Thrombopenia may 
be present in rats which are on a diet free from vitamin A 
before they show obvious signs of ill health. When a pro- 
found thrombopenia had been established the addition of 
vitamin A to the diet produced a rapid increase in the plate- 
lets to the normal number. Exposure to radium produced a 
lymphopenia, and also a thrombopenia if the dose was suffi- 
ciently large, but the animals recovered rapidly if the radium 
treatment was discontinued and the dose had not been too 
large. 

The views of Cramer and his co-workers have been con- 
traverted by Bedson and Zilva (1923), who report that rats 
fed diets deficient in vitamin A show a decrease in the number 
of platelets, but believe the decrease too small to be signifi- 
cant. Cramer, Drew, and Mottram maintain, however, that 
the technic of Bedson and Zilva was not sufficiently refined 
to justify confidence in their findings, to which Bedson and 
Zilva reply that on repeating the work with the same technic 
described by Cramer and his co-workers they were still un- 
able to confirm their observations. The relationship of 
deficiency of vitamin A to blood-platelet formation, there- 
fore, cannot be looked on as established. 

Origin.—It is certain that vitamin A is synthesized by 
flowering plants, for it is present in the leaves of all plants 
thus far examined. The source of vitamin A in fish oils has 
been definitely traced by Coward and Drummond to marine 


1500 LECTURES ON NUTRITION 


algee containing chlorophyl. Other lower marine plants dif- 
ferently adapted to photosynthesis (red weeds) are not so 
active in producing the vitamin, while those devoid of pig- 
ment which play a réle in carbon assimilation (mushroom) 
are almost completely deficient in it. 

Occurrence of Vitamin A.—Vitamin A is found most abun- 
dantly in certain foods of animal origin. Cod-liver oil appears 
to be the richest of all fats in this substance, but fish oils 
in general are good sources of it, as are also egg yolk fats and 
butter fat, the lipin extracts of glandular structures, such 
as liver, kidney, and testis. Vegetable oils, without excep- 
tion, have proved to be either deficient in vitamin A or to 
contain but small amounts of it. Among the vegetable 
products, leaves of plants, such as spinach, alfalfa, and celery 
leaves are rich in vitamin A, whereas those leaves which are 
thickened storage organs, such as cabbage and cauliflower, 
are more comparable to the tubers in that their content of 
vitamin A is low. An interesting correlation was proposed 
some years ago by Steenbock between yellow pigmentation 
in plants and vitamin A content. He demonstrated in the 
case of several products a striking relationship between yellow 
pigmentation and vitamin A content. Thus, he found white 
corn to be deficient, whereas yellow corn was moderately rich 
In vitamin A. Yellow turnips and sweet potatoes contain 
vitamin A, whereas colorless varieties do not. Red and 
blue corn did not contain the vitamin A unless yellow pig- 
mentation was also present. Red pigmented vegetables, 
such as the tomato, contain much yellow pigment masked 
by the red, and are correspondingly rich in the vitamin. 


OUR PRESENT KNOWLEDGE OF VITAMINS tor 


Palmer and Kennedy have shown clearly, however, that the 
relationship between vitamin content and yellow pigmenta- 
tion is a chance one. Pig liver is free from the carotin and 
xanthophyll pigments so widespread in the plant world, but 
is rich in vitamin A. The fats in the milk of certain species 
of animals, such as sheep, goat, swine, and rat, may be as 
white as lard, yet have a high content of vitamin A. Whereas 
the glandular organs of animals are rich in vitamin A, the 
muscle tissue is a very poor source of it. The body fats 
of animals may contain considerable vitamin A provided the 
food has been rich in this substance, or practically devoid 
of it if the vitamin was not provided in the ration. A number 
of investigators have shown clearly that the vitamin A is 
not present in milk fat unless it is supplied in the dietary. 

Properties —Miss Davis and I (1914) observed that the 
vitamin A was still present in yolks of hard-boiled eggs, which 
demonstrated a moderate degree of stability toward heat. 
We also demonstrated that butter fat could be saponified 
without destroying its vitamin A content. Osborne and 
Mendel passed live steam through butter fat for two and a 
half hours without producing marked deterioration in vita- 
min value. Steenbock, Boutwell, and Kent (1918) found that 
heating butter for four hours at 100° F. caused the destruc- 
tion of its vitamin A content. The reason for this was first 
pointed out by Hopkins, who showed that oxidation is a 
factor of prime importance in determining the rate of de- 
struction of the vitamin during the heating of foods. The 
vitamin A, therefore, is stable at high temperatures in the 
absence of oxygen. 


152 LECTURES ON NUTRITION 


Vitamin A is not extracted to an appreciable extent from 
plant products when the fats are removed by such solvents 
as ether, chloroform, benzene, or acetone. Hot alcohol is 
a much better solvent for its separation. Osborne and Men- 
del (1918, 1919) have shown, however, that ether or benzene 
extracts a certain amount of vitamin A from leaf structures, 
whereas water does not remove it. Emmett and Luros 
(1919) showed that benzene or acetone will not extract vita- 
min A from pancreas, thymus, and adrenals. Steenbock and 
Boutwell (1920) found that when carrots are saturated with 
lard or corn oil and then extracted with ether, little or none of 
the vitamin A is removed. Chloroform and carbon disulphid 
remove considerable amounts of vitamin A from carrots. 

Takahashi (1924, personal communication), in the lab- 
oratory of U. Suzuki, reports the isolation of vitamin A from 
cod-liver oil, butter fat, and green laver. He calls the sub- 
stance biostearin. It has the formula Co2.H.,O:. It is believed 
to contain two hydroxyl groups, one of which is tertiary, 
and the other primary or secondary. It is, therefore, an 
alcohol closely related to cholesterol. It is believed that 
cholesterol is a product of biostearin after it has undergone 
a physiologic change in the body. When 0.0001 per cent of 
biostearin is present in the diet of the rat, health and growth 
are maintained. He states that the effect of biostearin is pro- 
portional to its concentration in the diet up to 0.05 per cent, 
but when this amount is exceeded it has an unfavorable effect. 

Takahashi has prepared the benzoate, acetate, hexabromid, : 
and ozonid of the substance. It has a molecular weight of 
about 400. 


OUR PRESENT KNOWLEDGE OF VITAMINS 153 


When a small amount of biostearin was placed in a dish 
and covered with a dry plate the image of a screen placed 
between the plate and the biostearin appeared on the plate. 
This was true even when the plate was at a considerable 
distance from the biostearin. This happened even though 
the plate was carefully wrapped in black paper of the kind 
used to protect dry plates in commerce. When biostearin 
was sealed in a quartz tube and taken into a dark room, 
an image of an object between them was produced on a 
dry plate kept near the tube. The action of biostearin on 
the dry plate was much less when glass intervened than 
when quartz was used. 

When biostearin was placed in a flask and a current of 
carbon dioxid was passed over it and the gas was led out 
through an outlet before which a dry plate was held so that 
the gas which had been in contact with the biostearin was 
brought into contact with the plate no action was observed. 
When a dilute solution of biostearin was spread upon a dry 
plate and immediately washed off, the plate was not affected. 
When a dry plate was partly immersed in an alcoholic solu- 
tion of biostearin the submerged part was not affected, but 
the part of the plate outside of the solution was affected. 
Reduced copper or aluminum foil kept in the same vessel 
with biostearin was rapidly oxidized. In the environment 
of biostearin something is formed in the air which gives the 
KI-starch reaction and the paranitrophenylenediamin reac- 
tion. It is believed that it in some manner activates oxygen. 
Biostearin is a colored substance resembling the carotinoids, 
but unlike these it does not have a selective absorptive action 


154 LECTURES ON NUTRITION 


in the visible part of the spectrum. An alcoholic solution 
of the substance gave an absorption band near 3,200 A of 
the ultraviolet region. The solution becomes. fluorescent 
when illuminated with light from the iron arc. 

Requirements of Different Species for Vitamin A.—Defic- 
iency of vitamin A has produced ophthalmia in man, 
guinea-pig, rabbit, dog, swine, and chicken. Sugiura and 
Benedict (1922) drew the conclusion from experiments on 
pigeons that vitamin A is not essential in any stage of avian 
nutrition. They also found pigeons to remain in health 
on a diet lacking in vitamin C. 

Emmett and Peacock (1923, 1924) found that young 
chickens require vitamin A, but that young pigeons do not 
suffer from deprivation of this substance. It appears, there- 
fore, that the pigeon deviates from the general rule among 
birds in that either it does not require the vitamin A or is 
able to synthesize this substance. Beach (1923) described 
a pathologic condition in chickens caused by lack of vitamin 
A which would ordinarily be diagnosed as avian diphtheria 
or roup. ‘The disease was shown not to be infectious and 
could be controlled by giving foods rich in vitamin A. He 
described the development of xerophthalmia in chickens, 
and also found a remarkable accumulation of crystals of 
urates in the kidneys of chickens deprived of vitamin A. 
The kidneys were also pale and marked with a network of 
fine lines which are urate-filled tubules. White crystalline 
material, probably urates, was frequently noted on the liver 
and other organs. 


OUR PRESENT KNOWLEDGE OF VITAMINS 155 


VITAMIN B 

American and English investigators are apparently con- 
vinced that beri-beri is essentially a disease due to lack of 
vitamin B. In conversation with several eminent Japanese 
physicians I have found them still unwilling to admit that 
this is the sole cause of the disease. 

Walshe (1918) asserted that there are two factors in the 
production of beri-beri, the absence of a vitamin and the 
use of certain foods which are the direct and immediate 
cause of the disease. He suggests that in the absence of a 
specific vitamin carbohydrates undergo an aberrant  hy- 
drolysis with the production of toxic by-products, thus pro- 
ducing beri-beri. According to his view it is essentially an 
intoxication. Vedder (1923) suggests that beri-beri may be 
caused by the lack of two vitamins. In this way he would 
account for the two forms known as wet and dry beri-beri, 
McCarrison (1924) likewise insists that beri-beri is not directly 
a disease due to deficiency of vitamin B. He believes there 
are endemic areas in the Madras Presidency where people 
suffer from beri-beri on dietaries which would protect them 
in non-endemic areas. There can no longer be any question 
that experimental beri-beri or polyneuritis as produced in 
mammals in the laboratory is due to deficiency of vitamin B. 

Vitamin B is the most widely distributed of any of the 
known vitamins. It is present in all natural foodstuffs. 
Only manufactured products, as polished rice, white wheat 
flour, degerminated cornmeal, corn grits, and sugars, are 
essentially lacking in it. The vitamin B is more stable than 
either vitamin A or C. It appears to be an organic base and is 


156 LECTURES ON NUTRITION 


essentially insoluble in all solvents other than water, aqueous 
alcohol, and glacial acetic acid. Its isolation is attended 
with special difficulty because it occurs only in complex food- 
stuffs, and extracts which contain it are likewise contam- 
inated with very large amounts of numerous impurities. 
The substance occurs in but very small amounts in any 
foods. Yeast and the germ of wheat are two of the foods 
richest in this substance. 

Studies on Isolation of Vitamin B.—Williams (1917) ex- 
pressed the view that under certain conditions alpha-hydroxy- 
pyridin may possess antineuritic properties. He attributed 
these properties to the existence of this compound in the form 
of a pseudo-betain, and suggested that a configuration confirm- 
ing more or less closely to that of the betain ring was probably 
an essential characteristic of the vitamin B. It was pointed 
out that such a structure was possible in most of the simpler 
nitrogenous components of animal tissues, especially in the 
purin bases. Williams had previously brought forward the 
view that adenin may exist in a labile form in which it pos- 
sesses curative properties for polyneuritic pigeons. Dutcher 
(1919) asserts that desiccated thyroid, thyroxin, pilocarpin 
hydrochlorid, and other physiologic stimulants are able to 
induce ‘‘cures” with pigeons in polyneuritis. 

Besides alpha-hydroxypyridin, Williams has also stated 
that alpha-methylpyridone, trimethyl uracel, and 4-pheny]l- 
iso-cytocine gave slight protection. He considers vitamin B ~ 
to be a cyclic nitrogen compound with a substitution of oxy- 
gen in the ring and capable of existing in the betain con- 
figuration. Funk reported cures in the pigeon with hydan- 


OUR PRESENT KNOWLEDGE OF VITAMINS 157 


toin, and observed some curative properties in adenin and 
pyrimidin derivatives. 

Osborne and Wakeman (1919) described a method for 
preparing an extremely potent preparation of vitamin B. 
They prevented disintegration of yeast cells by boiling 
in acidified water after thorough washing. The protein was 
then coagulated and an extract, free from the products of 
autolysis, was obtained. The extracts from 4.5 kilos of fresh 
yeast, extracted with 15 kilos of water, were concentrated to 
2 liters and poured into 3 liters of 93 per cent alcohol. The 
precipitate which formed was inactive. The filtrate was 
evaporated and again poured into alcohol and the process 
was again repeated, so that a precipitate was finally obtained 
in an alcohol of 90 per cent strength. This amounted to 6.2 
per cent of the dry yeast and contained almost all of the vita- 
min B in the yeast. This preparation was not composed of 
pure vitamin, although it was extraordinarily active bio- 
logically. 

Shinza (1924) states that the symptoms of experimental 
polyneuritis are strongly like those due to depletion of the 
potassium in the body, and that the administration of potas- 
sium salts to polyneuritic birds had a restorative effect. 

Levene and van der Hoeven (1924) have prepared a more 
potent preparation of vitamin B than that secured by Os- 
borne and Wakeman. For the preliminary concentration 
of the material they employed the method of Osborne and 
Wakeman, then adsorbed the vitamin on silica gel. From 
this it can be extracted by alkalies and also by acids of pHs. 
They found that barium hydroxid incompletely precipitates 


158 LECTURES ON NUTRITION 


the vitamin from its solution, but a preparation was secured 
which they estimated to be 200 to 400 times as potent as 
that of Osborne and Wakeman. This material is obtained 
with very slight loss of vitamin. The preparation of Levene 
and his co-workers was active in daily doses containing 
0.00017 gm. of nitrogen per day. 

It was pointed out by McCollum and Simmonds (1918) 
that the pigeon test is uncertain, and that there is a high 
degree of improbability that so many totally unrelated chem- 
ical substances, as pyrimidins, purins, and pyridin, could 
fulfil the same physiologic purpose. They were led to sus- 
pect that the curative substances are not necessarily iden- 
tical with the indispensable nutritive principle essentia] 
for growth and normal functioning, but rather bodies which 
possess the pharmacologic properties of stimulating certain 
nerve cells to renewed activity. McCollum and Simmonds 
described a procedure for determining the presence or absence 
of the vitamin B. It can also be made roughly quantitative. 
The procedure is to restrict young rats to a diet of purified 
protein, dextrin, a salt mixture, and butter fat, or other fat 
which contains vitamin A. On such a diet the animals 
are unable to grow and ultimately die with or without the 
development of symptoms of polyneuritis. After from three 
to five weeks, while the animals are declining in weight, 
the substance to be tested is administered. If the animals 
recover and resume growth the test is positive. If they con- 
tinue to decline, the substance tested for is absent or present | 
in amounts too small to meet the nutritive needs. ‘This 
test requires considerable amounts of material. Only after 


OUR PRESENT KNOWLEDGE OF VITAMINS 159 


a week or two is the outcome apparent. Levene has adopted 
a modification of the growth test with the rat. In this test 
he employs rats about six weeks old and uses our basal diet 
for the production of polyneuritis. The animals are kept 
on this for three weeks. The average weight of his animals 
was 50 gm., and during the week immediately preceding the 
test they lost on the average 5 to 10 gm. The substance to 
be tested is given in solution. After from three to four days 
the animals are again weighed and the change in weight is 
accepted as an indication of the vitamin content of the 
solution. — 

Seidell (1924) has prepared very potent preparations of 
the vitamin B by taking advantage of the fact that Fuller’s 
earth exerts a selective adsorption for this substance as well 
as for alkaloids. From this “activated” Fuller’s earth the vi- 
tamin can be separated by treatment with barium hydroxid. 
From such concentrated preparation Seidell has prepared a 
silver compound and also a picrate of extraordinary potency 
when tested on pigeons. This picrate, although of extraor- 
dinary potency, is not a pure compound, since the crystalline 
product, of which 2 mg. daily protected pigeons, was not 
composed of homogeneous crystals, but of crystals of more 
than one kind. 

There is no convincing evidence that we know anything 
about the chemical nature of the vitamin B. We are not 
entirely certain that it is a single substance, and not several 
substances acting together. It is stable at high temperatures 
in acid solution, but in alkaline solution rapidly becomes 
denaturized. It is not destroyed by nitrous oxid, and so 


160 LECTURES ON NUTRITION 


appears not to be either a primary or secondary amin. Mc- 
Collum and Simmonds have tested a number of the sub- 
stances which are said by others to induce a cure of experi- 
mental polyneuritis in pigeons with the growth test in rats, 
and have invariably found that none of these exert the 
slightest beneficial effect on the polyneuritic rat. For this 
reason they have expressed skepticism concerning the speci- 
ficity of the pigeon test for the vitamin B. This raises the 
question as to whether or not the nutrient requirements of 
the pigeon and the rat with respect to this substance are iden- 
tical, a question which still remains to be decided. 

Relation of Histamin to Vitamin B.—Voegtlin and Myers 
(1919) called attention to the fact that vitamin B reacted 
to chemical reagents such as methyl alcohol, silver, lead, and 
barium salts in a similar way to secretin. Secretin prepara- 
tions from the duodenum of dogs relieved, to some extent, 
the antineuritic symptoms, and the vitamin B from brewer’s 
yeast on injection into dogs stimulated the pancreatic and 
biliary secretions. A yeast preparation which had lost its 
curative powers for avian polyneuritis was devoid of any 
stimulating effect on the pancreatic secretion and bile flow. 
Anrep and Drummond (1921) found that a yeast extract 
does not cause secretion of pancreatic juice as does secretin. 
Secretin can be extracted from the intestine of a cat showing 
polyneuritis. The suggestion of Voegtlin and Meyers that 
vitamin B and secretin are identical is not supported. 

Cowgill (1921) reports that intravenous injection of extracts — 
of rice polishings, of wheat embryo, of navy beans, of yeast, 
or of neutralized tomato juice, which all contain vitamin B, 


OUR PRESENT KNOWLEDGE OF VITAMINS 161 


does not stimulate the flow of saliva as does pilocarpin. 
Similar extracts were found by Cowgill and Mendel (1921) 
to be without noticeable effect on the rate of flow of pancreatic 
Juice, bile, or saliva in the dog. The intestinal mucosa of 
polyneuritic dogs was shown to contain secretin. No direct 
relationship has been established, therefore, between vitamin 
B and the secretory functions of the pancreas, liver, or salivary 
glands. 

Boyenval (1922) stated that on injecting histamin into rats 
fed polished rice he found no effects from the cachectic prog- 
ress of the disorder. On the other hand, the histamin-treated 
rats did not show the usual premortal nervous disturbances 
observed in the control animals. He thought histamin may 
exercise an antineuritic effect. Koskowski (1922) confirmed 
the findings of Boyenval. Although histamin stimulates the 
activity of the digestive glands, especially of the gastric glands, 
and thus aids in the rat’s digestion, it does not supply the 
lack of nitrogenous substances found in the pericarp of the 
rice grains and elsewhere, and it cannot, therefore, replace 
the antineuritic vitamin. Burge (1916, 1917), Dutcher 
(1920), and Stehle (1919) have presented conflicting observa- 
tions concerning the body content of catalase in polyneuritis 
and its possible réle in the pathology of that disease. Noth- 
ing definite has been established. Findlay (1921) reported 
a study of the effect of deprivation of pigeons of the vitamin 
B on the content of glyoxalase in their tissues. Glyoxalase 
is an enzyme which plays a réle in the metabolism of carbo- 
hydrate. It has the power to transform “glyoxals” into 


lactic acid. The “glyoxal”’ which serves in the animal body 
11 


162 LECTURES ON NUTRITION. 


as the intermediate substance between glucose and _ lactic 
acid is known as pyruvic aldehyde. Findlay found the 
amount of glyoxalase in the livers was reduced about one- 
half in polyneuritis. On inducing a “cure” by the admin- 
istration of the vitamin B the glyoxalase rose rapidly to two- 
thirds of the normal amount. 

Nature of Action of Vitamin B.—Green (1918) states that 
the daily requirement of pigeons for vitamin B varies with 
the exogenous metabolism. He believes there is no more 
reason to think that vitamin consumption is related to car- 
bohydrate metabolism than it is to protein or fat. Funk 
(1919) tried the effect of various substances which were known 
to influence carbohydrate metabolism, on the body sugar, 
liver glycogen, onset of beri-beri, loss of weight and length 
of life in normal pigeons and in others fed polished rice ex- 
clusively. The substances tried were dextrose, phloridzin, 
adrenalin, thyroid, parathyroid, and pituitrin. He con- 
cluded that the antiberi-beri vitamin played an important 
role in carbohydrate metabolism. Vedder has, however, given 
experimental evidence which is opposed to this view. 

Dutcher (1918) was of the opinion that the carbohydrate 
effect was due to an overloading of the oxidative mechanism 
of the body rather than to a specific relation between the 
metabolism of carbohydrate and vitamins, the latter being 
used up in the process. He believes that in specific fasting 
for vitamin B there is an accumulation of incompletely met- 
abolized products which affects the nervous system and ac- 
counts for its loss of function. He further stated (1920) 


that the antineuritic substance functions as a metabolic 


OUR PRESENT KNOWLEDGE OF VITAMINS 163 


stimulant, since the body temperature fell during the de- 
velopment of avian polyneuritis and rose after giving vita- 
min B. He observed the catalase content of the tissues of 
-birds suffering from the disease to be decreased to 56 per cent 
of the normal. It returns to normal when vitamin B is ad- 
ministered. He interpreted his results as indicating a reduc- 
tion of the oxidation processes in polyneuritis. Such a de- 
pression of oxidation results, he believed, in the accumula- 
tion of toxic metabolism products which affect the nervous 
system. 

Karr (1920) studied the effect of specific fasting for the 
vitamin B on the desire of dogs to take food. He fed diets 
composed of isolated food substances and especially free 
from vitamins A,.B, and C. The dogs were restricted to 
this diet until they refused to take the food, and then a source 
of vitamin B was fed separately. Yeast, milk, tomato, anda 
concentrated extract of vitamin B were employed for this 
purpose. The addition of these substances resulted in prompt 
response of the appetite. Karr concluded that there was 
some relation between the desire for food and the amount of 
vitamin B ingested. Brewer’s yeast was much more effective 
than baker’s yeast for this purpose. He also studied the 
metabolism of dogs deprived of vitamin B by making quan- 
titative studies of the assimilation of nitrogen. No decrease 
in the capacity for digestion nor in the character of the 
metabolic products eliminated could be detected as the 
result of specific starvation for this substance. He found 
that vitamin A had no such effect in influencing the appetite 
as did vitamin B. Cowgill (1921) confirms Karr’s view that 


164 LECTURES ON NUTRITION 


“there is some relationship in dogs between the desire to 
partake of food and the amount of so-called water-soluble 
vitamin ingested.””’ Kennedy and Dutcher (1922), on the 
other hand, insist that the effect of vitamins is not necessarily 
one of body stimulation, but rather a stimulation of the 
metabolic processes which promote growth or normal func- 
tioning. Theirs appears to be the more logical view. 
Pathology of Beri-bert.—A new conception of the pathology 
of polyneuritis and other deficiency diseases was introduced 
by McCarrison (1919). Loss of the co-ordinating powers 
of the muscles is the most striking feature of polyneuritis. 
The onset of the disease is generally preceded by the bird 
sitting with ruffled feathers and with the appearance of ill- 
ness. There is progressive weakness, and when disturbed 
there is a tendency for many pigeons to be taken with con- 
vulsive seizures in which they turn ‘“‘cart-wheels” backwards 
at intervals. In the acute type of the disease many birds 
sit with the head greatly retracted. These symptoms gen- 
erally led investigators to accept the view that the lesions 
in beri-beri were principally situated in the nerve system. 
McCarrison has presented evidence that injury to the nerves 
is much less pronounced than injury to certain other tissues. 
He observed functional and degenerative changes in the thy- 
mus, testicles, spleen, ovary, pancreas, heart, liver, kidneys, 
stomach, thyroid and brain, atrophy being apparent in every 
case, the severity being in the order named. The adrenals, 
on the other hand, suffered hypertrophy. ‘This was asso- 
ciated with a proportionate increase in the content of the 
glands in adrenalin. Edema was invariably associated with 


OUR PRESENT KNOWLEDGE OF VITAMINS 165 


hypertrophy of the adrenal glands, suggesting its relation 
to an excessive production of adrenalin. Inanition gave rise 
to adrenal hypertrophy and atrophy of other organs, the 
brain excepted, similar to that observed in birds fed solely 
on polished rice. Rice is deficient in all known vitamins, 
protein, and in several inorganic elements. The gastric, 
intestinal, biliary, and pancreatic disorders of birds fed only 
polished rice were more serious than the nerve lesions. This 
diet gave rise to congestive and atrophic changes in all the 
coats of the intestine, especially the duodenum; to lesions 
in its neuromuscular mechanism; to impairment of its diges- 
tive and assimilative functions; and to failure of its pro- 
tective resources against bacterial infection. Guinea-pigs 
restricted to a diet of oats and autoclaved milk developed 
lesions of the digestive tract analogous to those seen in pig- 
eons fed polished rice. Owing to the multiple deficiencies 
of the rice diet it is not possible to decide as to the specific 
effects of deficiency or lack of a single dietary component. 
It is interesting to note, however, that on a diet deficient 
only in vitamin C, guinea-pigs developed lesions comparable 
with those in birds with diets faulty in several respects. 
McCarrison fed pigeons a diet of polished rice, butter fat, 
and raw onions. Typical polyneuritis developed. In these 
birds atrophy of the myenteron and of the elements of the 
mucosa were often comparatively slight. The results of this 
experiment are very significant for the interpretation of 
much of McCarrison’s data, for with a diet of polished rice, 
butter fat, and onions the most pronounced deficiency was 
in the antineuritic principle. Certain inorganic elements, 


166 LECTURES ON NUTRITION 


especially calcium, phosphorus, and potassium, were supplied 
in very inadequate amounts, and the intestinal lesions pro- 
duced were less pronounced than with a diet of polished 
rice alone. The addition of vitamin A in butter fat and 
of vitamin C in onions apparently improved the well-being 
of the birds to a marked degree. The confusion in this field 
is well illustrated by mentioning again in this connection 
that certain investigators believe that the pigeon requires 
neither vitamin A nor C. 

In a number of McCarrison’s polyneuritic birds the blood 
was found to be infected by Bacillus suipestifer, Bacillus pyo- 
cyaneus, and another organism not identified. He emphasized 
his belief that systemic infection is rendered easier by the 
presence of the pathologic processes existing in the intestine 
as the result of dietary deficiencies. Such invasion was 
favored by the impaired production of digestive juices by 
the malnutrition of the secretory cells owing to the con- 
tinued congestion of the mucous membrane of the intestine 
and the blood, by the greater opportunity which the de- 
bilitated mucous membrane provided for the growth of micro- 
organisms on its surface, and to actual breaches of continuity 
in the walls of the bowel itself. The imperfect digestion 
in the upper part of the tract offered a favorable medium 
for the growth of bacteria. Unwholesome products, he 
believes, tend further to debilitate the mucosa and to in- 
crease the prospects of invasion by micro-organisms. In- 
fection of the blood from the bowel under such conditions 
would be expected and was repeatedly demonstrated by 
aérobic culture of the heart blood.. It must be emphasized 


OUR PRESENT KNOWLEDGE OF VITAMINS 167 


in this connection that in no instance did McCarrison feed 
a diet complete except for a lack of the vitamin B, and so it 
is not yet possible to determine which of the pathologic 
effects that he observed were the result of vitamin B de- 
ficiency and which were caused by other deficiencies in his 
experimental diet. 

Cramer, Drew, and Mottram (1921) observed that the ab- 
sence of vitamin B from the diet of mice and of rats led to 
atrophy of lymphoid tissue throughout the body, and also 
to lymphopenia in the circulating blood. ‘The number of 
polymorphonuclear leukocytes was not affected. The ab- 
sence of vitamin A did not lead to atrophy of the lymphoid 
tissue and there was no lymphopenia. They found that 
absence of the vitamin B led to characteristic nutritional dis- 
turbance, such as loss of weight, emaciation, subnormal tem- 
perature which may be designated by the term “marasmus.” 
No such marasmic condition resulted from a lack of vitamin 
A. This condition can be produced by other processes which 
destroy lymphocytes, such as x-ray exposure. They con- 
clude that vitamin B is necessary for the normal function- 
ing of the lymphoid tissue. These investigators (1922) made 
a histochemical study of fat absorption from the intestine. 
They demonstrated that the functional activity of the in- 
testinal epithelium as regards absorption is profoundly 
affected by the presence in the food of vitamins A and B, 
particularly the latter. They believe that the vitamins have 
a stimulating action on absorption and probably also on in- 
testinal digestion. In the absence of vitamins they found no 
delay in the passage of food in such animals. After exposure 


168 LECTURES ON NUTRITION 


to radium sufficient to produce lymphopenia the absorption 
of food was impaired in the same way as if vitamins A and 
B were absent, and this effect could not be counteracted by 
an abundant supply of vitamins in the food. These observa- 
tions confirm the view previously expressed by the authors 
that vitamins play an important part in the absorption of 
food, and the marasmus resulting from a deficiency of vitamin 
B is due to an impaired assimilation from the intestine. 
Findlay (1923) observed that diets lacking in vitamin B 
produce in pigeons and rats definite changes in the hemato- 
poietic tissues, consisting of congestion and hemorrhage in 
the bone-marrow, followed in the more chronic cases by 
gelatinous degeneration. He noted that pigeons fed a diet 
lacking in vitamin B were much more susceptible to infec- 
tion with pneumococcus and meningococcus, organisms to 
which they are naturally quite immune. They were also 
much more susceptible to Bacillus colt and Bacillus enteritidis. 
The reduction of natural immunity he found to be related to 
a fall in body temperature produced by diets deficient in 
vitamin B. It was only marked when the cloacal temperature 
was lowered to 40° C. or below. He stated that the lowering 
of the body temperature appeared to decrease the resistance 
by (a) facilitating the growth of the invading organisms, 
(b) reducing the leukocytic response to the infection, and (c) 
reducing the bactericidal power of the leukocytic exudate. 
Very few investigators, even among biochemists, who are 
best in a position to understand the interpretation of quality | 
in diet, are yet able to plan and prepare diets which are 
complete in every respect except for a single missing factor. 


OUR PRESENT KNOWLEDGE OF VITAMINS 169 


Bacteriologists, immunologists, and others are in many cases 
wholly unprepared to manage the dietary phase of their 
studies designed to show the pathologic effects of diets faulty 
in specific ways. ‘The result is that much of the literature 
deals with experiments in which multiple defects were char- 
acteristic of the dietaries, and so interpretation is not possible. 
That very important discoveries are still to be made in the 
pathology of malnutrition there can be no doubt, but much 
more work and better work is necessary before these dis- 
coveries can be made. A recent observation by Webster and 
Pritchard (1924) is of special interest. They report that white 
mice from the Rockefeller Institute breeding room fed: on 
the McCollum-Simmonds diet, consisting of whole wheat 
67.5, casein 15, milk powder 10, sodium chlorid 1.0, cal- 
cium carbonate 1.5, and butter fat 5.0 per cent, are more re- 
sistant to typhoid infection, mercury bichlorid intoxication, 
and botulinus toxin than are similar mice fed on bread and 
pasteurized milk supplemented by an oatmeal and buck- 
wheat mixture and dog biscuit. This comprehensive study 
emphasizes the necessity of feeding diet exactly controlled 
in every detail if one would secure optimal as contrasted with 
good nutrition; and it indicates that the body profits in a 
physiologic sense by an optimally constituted diet in the same 
degree that success is achieved in chemical operations follow- 
ing the best procedure. 


BIOS AND THE GROWTH OF YEAST 


Investigators have been very desirous of isolating and iden- 
tifying vitamin B. We have as yet no qualitative tests which 


170 LECTURES ON NUTRITION 


characterize it, and accordingly any effort at separating this 
interesting substance must be controlled by feeding tests 
on every fraction or solution which appears of interest. 
Such a method is time consuming and requires an extravagant 
expenditure of material. As a result of this Williams sought 
to use yeast as a test organism for determining the content 
of vitamin B in extracts of natural foods. It was observed 
by Wildiers (1901) that yeast grew very slowly in a medium 
of pure sugar and inorganic salts where ammonium sulphate 
was the only source of nitrogen, unless the seeding was large. 
Pasteur had already observed that single yeast cells did 
not proliferate in a medium of relatively pure substances as 
did seedings containing many cells. Wildiers sought to ex- 
plain this phenomenon by postulating the existence of a special 
nutrient principle necessary for yeast proliferation to which 
he gave the name “bios.” Williams confirmed Wildiers’ 
findings that extracts of certain substances, such as yeast 
cells, greatly stimulate the rate of proliferation of yeast. 
Williams was impressed by the fact that those substances 
which are richest in vitamin B gave extracts which were 
most potent in stimulating yeast growth, and conceived the 
idea that the stimulating substance was the vitamin B. 
Williams devised a procedure by means of which the stimulat- 
ing effects of various extracts and various amounts of these 
on the proliferation of yeast cells during a period of eighteen 
hours could be readily determined by counting the progeny 
of a single cell produced during this interval. | 

Souza and McCollum sought to apply the method of Wil- 
liams and discovered that the test was not a test for the vita- 


OUR PRESENT KNOWLEDGE OF VITAMINS 171 


min B, but for some other substance which affects yeast. 
Extracts of beef muscle or of rolled oats stimulate the growth 
of yeast in a purified medium, but the same is true of these 
substances made after alkalinizing the preparation and heat- 
ing to a point where growth tests on rats indicate that the 
vitamin B has been destroyed. Obviously bios has a stability 
in the presence of alkali not possessed by vitamin B. 

Nelson, Fulmer, and Cessna (1921) demonstrated that 
yeast growing in a purified nutrient solution at a slow rate 
synthesizes vitamin B. This was confirmed by MacDonald. 

Utilizing this slow growth of yeast, MacDonald accumu- 
lated 6 to 14 gm. of dry yeast cells from several strains or 
pure organisms, transplanted at weekly intervals to fresh 
nutrient medium free from either bios or vitamin B. Such 
yeast when fed to rats declining in. weight for lack of vitamin 
B led to a response entirely comparable to what one sees in 
such experiments when similar amounts of commercial 
yeast are added. 

Fulmer and Nelson used a synthetic sugar, methose, pre- 
pared from formaldehyd, as the sole source of energy in 
a medium in which yeast was grown, and in the absence of 
both bios and vitamin B. Under these circumstances yeast 
can undergo cell proliferation which is greatly stimulated 
by the addition of the substance bios. The work of several 
investigators has been adversely criticized because of the 
assumption that the cane sugar employed in the medium 
still contained traces of vitamin B (bios). 

Numerous studies have been made during the last four 
years on the effect of various preparations of yeast prolifera- 


172 LECTURES ON NUTRITION 


tion with a view to throwing some light upon the nature of 
bios. Fulmer and co-workers (1924) believe they have 
demonstrated that bios is not a single substance, since the 
combination of two extracts, neither alone very active, was 
much more effective. 

Eddy, Kerr, and Williams (1924) have isolated from yeast 
a crystalline substance melting sharply at 223° C. They be- 
lieve this substance to be a bios. ‘The recrystallized product 
retains its melting-point and shows no loss of activity. The 
yield of the product was about 0.03 per cent of the dry yeast. 
It contains 43.29 per cent of carbon and 8.31 per cent of 
hydrogen. ‘The substance gives no ninhydrin or biuret 
reaction. This substance, they say, stimulates the growth 
of yeast. Their crystalline preparations show but a single 
crystal form under the microscope. They assign the provis- 
ional formula C;Hi,NO;:, admitting possible variation in 
the hydrogen value. Notwithstanding this seeming con- 
clusive evidence of the individual nature of the substance 
isolated, and its stimulating effect on yeast, Eddy and co- 
workers state: “‘Nothing in our method of isolation negates 
the possibility of more than one bios. In fact, the relative 
stimulation producible by the addition of our pure product 
and by the use of autolyzed (yeast) lends strong probability 
to the suggestion that the latter contains more than one 
growth stimulant.” Miller (1924) and later Deas (1924) 
have presented extensive experiments strongly suggesting 
that there are at least two bioses. | 


OUR PRESENT KNOWLEDGE OF VITAMINS 173 


VITAMIN C 

There appears to be unanimity of opinion among all in- 
vestigators that scurvy is a condition resulting specifically 
from a lack of the vitamin C. Modern knowledge of the 
etiology of scurvy dates from a discovery by Holst and Frohlich 
(1912) that guinea-pigs rapidly develop the disease when 
confined to a diet of cereals or of bread. They observed 
that excessive feeding of guinea-pigs with carrots, turnips, 
or dandelions did not lead to the development of scurvy, 
although the animals on this food suffered considerable 
loss in weight. The addition of small amounts of fresh cab- 
bage or carrots or other fresh vegetables cured animals 
suffering from scurvy. They showed that the antiscorbutic 
substance is destroyed by cooking or drying. It was defi- 
nitely shown by Holst and Frdéhlich (1922) that the anti- 
scorbutic potency of fruits and vegetables displayed a greater 
stability in acid than in alkaline solution. They observed 
that fruit juices were more heat stable than vegetable juices, 
and suggested that the acidity protected the antiscorbutic 
principle. Fiirst (1912) found that whereas dry cereals or 
pulse did not prevent scurvy, they acquired antiscorbutic 
properties when allowed to germinate. 

Owing to the ease with which the vitamin C is destroyed 
in ordinary cooking or drying of fruits, vegetables, milk, and 
so forth, much interest has been manifested by several inves- 
tigators in the study of the vitamin potency of various natural 
foods in the untreated condition and when subjected to vary- 
ing degrees of heat treatment. Special interest has centered 
on the vitamin content of fresh milk, powdered milk, and pas- 


174 LECTURES ON NUTRITION 


teurized milk. There seems to be no doubt that pasteurized 
milk is nearly devoid of vitamin C, but it is asserted that 
certain milk powders still retain a demonstrable amount of 
this substance. It has been conclusively shown by Dutcher 
and co-workers (1920) and by Hart and co-workers (1919) 
that the vitamin C content of milk is dependent on the food 
of the cow, so that summer milks from cows on green feed 
are much richer in vitamin C than winter milks. Since there 
is such great variation in the vitamin C content of fresh milk, 
it hardly seems worth while to emphasize to the extent that 
has been done the importance of preserving the antiscorbutic 
properties of milk. It seems best to look to other articles in 
the diet to supply this principle. 

Vitamin C is present in all fresh fruits and fresh vege- 
tables. The tomato and the citrous fruits appear to be rich- 
est in it, and apples, grapes, pears, peaches, and other of our 
commoner fruits appear to be among the poorest of the fresh 
foods’in vitamin C. Cabbage, lettuce, celery, and carrots 
are all excellent sources of it. 

This is the most unstable of the vitamins. It is especially 
sensitive to oxidation, but recent investigators have demon- 
strated that it is much more stable at high temperatures in 
the absence of oxygen than was formerly supposed. 

A most interesting observation with respect to vitamin C 
was made by Kohman and Eddy. They have apparently 
solved the problem of canning fruits and vegetables so as 
to preserve the vitamin C content. Taking advantage of 
the knowledge that fruits and vegetables when harvested 
are living and respiring structures, they (1924) have so treated 


OUR PRESENT KNOWLEDGE OF VITAMINS 175 


these articles as to use up the oxygen in the tissues before 
applying heat for the canning process. Apples contain 
about 5 per cent by volume of oxygen, and, as in other fruits, 
the process of respiration goes on. When the supply of 
oxygen is cut off the oxygen in the apple is used up in ap- 
proximately thirteen hours. The apples are prepared for 
canning by peeling and quartering, and are then covered 
with a weak solution of salt in water to prevent access of air. 
The oxygen in the fruit is rapidly consumed and the fruit 
can then be canned or cooked in any way which seems de- 
sirable without loss of vitamin C. Apples prepared in this 
way and stored for nine months showed no appreciable 
loss of vitamin C. Since apples when held in cold storage 
gradually lose their vitamin C content, canning by this 
method actually preserves the vitamin much better than when 
the fruit is kept in its natural condition. Canned spinach 
nine months after canning was shown to be one of the richest 
sources of vitamin C yet found. They report that feeding 
tests with cabbage processed by a commerical method of 
canning showed a loss of vitamin C which is markedly less 
than that of cabbage cooked in open kettles. The loss is 
due to oxidation. Temperature and time of heating were 
not found to be factors. 

Bezssonov (1922) is the only investigator who has yet 
attempted to prepare concentrated preparations of the vita- 
min C. He subjected cabbage to hydraulic pressure and 
immediately treated the expressed liquid with neutral lead 
acetate. The filtrate from this precipitate was evaporated 
to dryness under diminished pressure at 35° F. From each 


176 LECTURES ON NUTRITION 


100 c.c. of the juice he obtained 2.5 gm. of a slightly yellowish 
hygroscopic powder which contained 33 to 45 per cent of 
reducing sugar and 52 to 65 per cent of total sugar, as 
well as about 7.5 per cent of ash. This substance, free 
from fats and proteins, was active in protecting guinea- 
pigs in daily doses of 0.1 gm. Zilva (1923, 1924) has 
shown that the sugars can be removed from desiccated 
lemon juice by fermentation with yeast without destroying 
vitamin C. 

Anatomic Lesions in Scurvy—The two most noticeable 
conditions observable at necropsy are hemorrhage and frag- 
ility of the bones. The stomach, intestines, and cecum may 
show congestion, hemorrhage, or ulceration, but, according 
to Cohen and Mendel, these organs may in some cases be 
normal in appearance. These pathologic conditions are 
usually observed in animals fed oats and water or oats and 
milk, and less frequently in those fed the experimental diet 
of Cohen and Mendel. It is pointed out that an ever-present 
condition in advanced scurvy is lack of appetite. The food 
intake diminishes as the disease advances, but fasting does 
not produce the characteristic lesions of scurvy. 

A remarkable observation made by Cohen and Mendel 
(1918) is that the swollen joints and tenderness often appear 
while the animals are still growing rapidly and have good 
appetites. Inanition can, therefore, play but a minor réle, 
if any, in the production of the more prominent symptoms of 
scurvy. There seems to be regularly a period of ten days 
or so of fasting previous to the death of the guinea-pigs which 
are confined to a scorbutic diet of soy beans or cereals, for 


OUR PRESENT KNOWLEDGE OF VITAMINS 177 


the loss of weight during this period corresponds to the rate 
noticed in guinea-pigs during starvation. 

LaMer and Campbell (1920) found an increase in the weight 
of the adrenal glands which they believe may represent a 
compensatory response to the decreased adrenalin production 
known to exist in the scorbutic animal. They believe that 
the heart and kidneys of scorbutic animals increase in weight, 
but that the liver is not enlarged. | 

Morel and co-workers (1921) state that the bone changes 
seen In experimental scurvy are not related to defects in cal- 
cium metabolism. Morikawa (1920) observed the following 
changes in the suprarenals in experimental scurvy in guinea- 
pigs: increase in weight, increase in lipoidal content of the 
cortex, and reduction in amount of doubly refracting fat. 
He states that the lipoid in the zona fasciculata was distributed 
in three layers; an outer layer rich in lipoids, a middle layer 
poor in lipoids, and an inner layer rich in lipoids. McCar- 
rison (1921) has reported a remarkable enlargement of the 
suprarenal glands in the guinea-pig during experimental 
scurvy. The changes in these organs were slight during the 
first fifteen days. During this periqd the weight of the spleen, 
liver, stomach, and intestines was definitely reduced. These 
results have been confirmed by others. Wells (1921) says 
that the tooth pulp is abnormal in scorbutic guinea-pigs. 
Hess, Unger, and Pappenheimer (1922) found that prolonged 
exposure to direct light did not prevent scurvy in guinea-pigs. 
Gerstenberger and Burhans (1922) found that scorbutic 
infants and guinea-pigs, as well as polyneuritic pigeons, 


burned carbohydrates completely. Iwabuchi (1922) found 
12 


178 LECTURES ON NUTRITION 


a decrease in the amount of ash in muscle and bone from 
scorbutic guinea-pigs. The fat content of the muscles and 
adrenals was decreased. The calcium content of the blood 
was 50 per cent below normal. Refractometric readings 
showed the protein content of the blood to be abnormal. 
Hemoglobin was markedly decreased, although the number of 
red corpuscles was essentially normal. Iwaduchi, however, 
could not confirm the observations of others that the cal- 
cium and phosphorus content of the blood was abnormal. 
Smith (1923) found no evidence of the deposition of calcium 
in the tubercle in infected guinea-pigs when calcium was 
administered together with cod-liver oil. He states that cod- 
liver oil has a definite but slight effect on the nutrition of 
the non-tuberculous guinea-pig. 

Liotta (1923) found that there was a marked diminution 
in the number of red corpuscles, as well as in the amount of 
hemoglobin, and a slight increase in the number of leukocytes 
of the blood of guinea-pigs in scurvy produced by a diet com- 
posed exclusively of oats.. 

Hojer (1924) has discussed at length the pathology of the 
bones, teeth, muscles, heart, liver, spleen, kidney, adrenal 
and salivary glands, lung, blood, blood-vessels, and connective 
tissue in scurvy in guinea-pigs. 

It is not easy to evaluate these observations because the 
guinea-pig is such an unsatisfactory animal with which to 
conduct any sort of experimental study. The diets employed 
to produce experimental scurvy in guinea-pigs have almost 
always been unsatisfactory in some respects other than a 
deficiency of vitamin C, and so one must qualify any state- 


OUR PRESENT KNOWLEDGE OF VITAMINS 179 


ments respecting the pathologic lesions resulting from de- 
privation for vitamin C, since it is not certain that other 
factors may not have contributed to produce these lesions. 

For the benefit of those who are accustomed to regard 
an individual as well until clinical recognizable symptoms are 
apparent, it is appropriate to mention that Dr. James Lind, 
who in 1747 wrote his classic account of scurvy, in discussing 
scurvy in sailors made an observation which deserves our 
careful consideration today in relation to the effect which 
the diet may exert on the general well-being and behavior. 
He says: “An uncommon degree of sloth and laziness which 
constantly accompanies this evil (scurvy) is often mistaken 
for the wilful effect of the patient’s disposition. This may 
prove fatal to many, some of whom when obliged by their 
officers to climb up the shrouds have been seen to expire 
and fall down from the top of the mast.” 

The cause of scurvy is now so well understood that there 
is little reason why it should ever attack masses of people 
except under extraordinary conditions of war and famine. 
The prevention of scurvy in infants is, however, a matter of 
importance, since the infant. fed solely on heated milk sup- 
plemented with any list of foods not including fresh, unheated 
fruit juices, is likely to develop the disease within a few weeks. 
There are many still who do not recognize it. The wisest 
policy is to examine the diet of the child to see whether it has 
the antiscorbutic principle rather than to rely on clinical 
observations to determine whether it is in special need of 
vitamin C. The safe course is to give every child a suitable 
antiscorbutic fruit juice, such as orange juice or tomato juice. 


180 LECTURES ON NUTRITION 


It has been definitely shown that man, monkeys, and guinea- 
pigs develop scurvy when deprived of vitamin C, but it is 
certain that the rat, prairie dog, and apparently also birds 
are immune to it. This immunity has been shown in the case 
of the rat to be due to an ability on the part of this species to 
produce synthetically the vitamin C. This is demonstrated 
by an abundance of vitamin C in the livers of animals which 
have been deprived of the vitamin during the entire period 
of their growth. Such livers are highly effective in the cure 
of acute scurvy in guinea-pigs. 


VITAMIN D 


The experimental studies on rickets during the last few 
years constitute the best illustration which nutrition studies 
have yet produced of the manner in which animal experi- 
mentation can solve certain medical problems. Once a 
pathologic condition is produced experimentally in an ani- 
mal, either by accident or design, if it be due to defects in 
the dietary, the solution of the problem and the correction 
of the dietary fault can be undertaken with the certainty 
that success will be the reward of effort. This follows from 
the completeness of our present-day knowledge of the essen- 
tials of a complete diet and of the great body of dietary in- 
formation which we possess concerning the quality of many 
natural foodstuffs with respect to each of the essential 
nutrient principles. Experimental diets can now be planned 
which have any defect or defects desired, and which are 
known with reasonable certainty to be complete in all other 
respects. 


OUR PRESENT KNOWLEDGE OF VITAMINS 181 


Glisson (1650) published the first adequate description 
of a disease which is still commonly known as rickets. The 
condition had appeared in parts of England about thirty 
years previous to his writing. Later Barlow (1894) described 
the relationship of infantile scurvy and rickets. 

Rickets is a disease which affects the entire body, although 
the most noticeable signs of it are seen in the bones. At the 
beginning of the disease the children are usually constipated. 
They are restless and irritable, usually apathetic and dis- 
inclined to play. They sleep poorly. Frequently a rachitic 
child rolls its head about on the pillow until the hair is worn 
off from the back of its head. The child perspires freely, 
especially about the head. 

The muscles are lax and the tendons and ligaments may be- 
come elongated. Because of this, as well as from the softening 
of the bones, children do not walk or stand at the proper 
time. They do not profit by their opportunity for exercise 
like the normal child. The muscles of the intestine are 
affected as well as those of the legs and arms. Owing to weak- 
ness of the intestinal musculature and the muscles of the 
abdomen a pot belly develops. 

As the disease advances deformities of the bones begin to 
appear. One of the first of these is the ‘“‘rachitic rosary” or 
a line of knobs on the side of the chest where the bones of 
the ribs join the cartilages. The walls of the chest are drawn 
inward every time the child breathes, and grooves are formed 
along either side of the line of attachment of the diaphragm. 
The chest becomes compressed from side to side and “‘pigeon 
breast”’ deformities develop. Bosses of new bone are formed 


182 LECTURES ON NUTRITION 


on the side and front of the skull, and the head acquires a 
square shape. The ends of the long bones of the extremities 
become enlarged. The legs become knock-kneed or bowed. 
The bones of the arms bend, and there is marked enlarge- 
ment of the epiphyses at the wrists and ankles. In severe 
cases curvatures of all sorts appear. Some children show very 
severe anemia. Some manifest extreme nervousness and often 
convulsions. Rickets is seldom fatal, but often the child dies 
from some complication, especially bronchopneumonia. 

Rickets is in great measure confined to civilized races and 
to the temperate zone. The reason for this has long been a 
puzzle. Wild animals almost never suffer from rickets, and 
we now know that under domestication rickets is always 
associated with defective diet. 

Experimental Rickets—Mellanby (1919) produced condi- 
tions which he believed to be rickets by confining puppies 
to a considerable number of faulty diets. He did an im- 
mense amount of work, but apparently was unaware of the 
various factors which are essential for an adequate diet. 
His experimental work was not planned with a definite under- 
standing of the dietary properties of the foodstuffs which he 
used. ‘This is evident from statements like the following: 
“The greater the amount of bread eaten, the greater will be 
the tendency to rickets.”’ “Whether, therefore, the protein 
effect depends on its own action remains to be decided. 
If the antirachitic effect of protein is established we will be 
able to comprehend one reason why milk is a better preven- 
tive of rickets than the corresponding amount of butter.” 

Mellanby was, however, the first to associate a group of 


OUR PRESENT KNOWLEDGE OF VITAMINS 183 


fats rich in vitamin A with the prevention of rickets. Whereas 
some of his data were suggestive that vitamin A was essential 
for bone growth, some of his results indicated that perhaps 
another factor was needed which was not identical with 
vitamin A. His experimental work did not prove or disprove 
this point. This is true for the reason that in many cases 
Mellanby did not appreciate the importance of the calcium 
to phosphorus ratio in his study of rickets. His experiments 
brought to light the following facts: Cod-liver oil possesses 
great antirachitic potency; suet and butter fats are also effec- 
tive in producing calcification, whereas lard has no rachitic 
effect; peanut oil is the most effective of the vegetable oils, 
Cod-liver oil was found to be much superior to butter fat in 
preventing rickets. 

The therapeutic value of cod-liver oil in rickets had long 
been accepted by many, but the nature of the curative sub- 
stance had not yet been recognized. It is not surprising that 
Mellanby emphasized the fact that those fats especially 
potent in preventing rickets contained a high content of the 
vitamin A, and he was led to associate a lack of this vitamin 
with the disease. 

Although Mellanby’s studies are open to obvious criticism, 
nevertheless his experiments constituted pioneer work. 
Since the dietary requirements of the dog are not satisfac- 
torily worked out, it is doubtful whether any one could have 
planned experimental diets which were as clean cut and de- 
cisive as those which have been used with the rat. 

When Mellanby’s studies were reported, a co-operative 
investigation between the Department of Pediatrics of the 


184 LECTURES ON NUTRITION 


Johns Hopkins Hospital and the Department of Chemical 
Hygiene of the School of Hygiene and Public Health, Johns 
Hopkins University, was in progress. Our program included 
a detailed study of the effects of diets faulty in specific ways 
on the growth and structure of the bones, and also studies 
on the composition of the blood with respect to its inorganic 
elements. 

A notable advance in our knowledge of the etiology of 
rickets came in the spring of 1921. In March of that year 
Sherman and Pappenheimer published an important paper 
demonstrating that rickets could be produced in rats on a 
diet low in phosphorus, and that it could be prevented by 
the addition of alkaline potassium phosphate. Shipley, 
Park, McCollum, and Simmonds reported in May similar 
experiments. ‘They reported the production of rickets by 
means of two diets. Both diets were deficient in vitamin A 
and phosphorus. When a complete salt mixture replaced 
the sodium chlorid and calcium carbonate the diets no longer 
produced rickets, but typical osteoporosis. ‘These observa- 
tions showed the importance of the inorganic constituents 
of the diet, especially of phosphorus, in relation to the disease. 
With the exception of Mellanby, who had placed but little 
importance on the salt composition of the diet, almost all 
other investigators have sought to produce rickets by a re- 
duction in the calcium. 

Our experience has shown that a diet of cereals and legu-_ 
minous seeds, with casein 10 per cent and calcium carbonate 
3 per cent, induced rickets in rats. We fed another group 
of animals this diet, but increased the casein to 20 per cent, 


OUR PRESENT KNOWLEDGE OF VITAMINS 185 


leaving the calcium carbonate at 3 per cent. These animals 
grew well and had normal bones. We had at this time made 
the observation, as had also Sherman and Pappenheimer, 
that the phosphate ion under certain conditions would pro- 
tect against rickets. Since casein is a phosphorus-containing 
protein, we decided to replace it in this diet by the non- 
phosphorized proteins gelatin and wheat gluten, leaving 
3 per cent of calcium carbonate in the diet as before. Diet 
3,143 has the following composition: wheat, 33.0; maize, 33.0; 
gelatin, 15.0; wheat gluten, 15.0; sodium chlorid, 1.0; and 
calcium carbonate, 3.0 per cent. Since the diet contained 
considerable amounts of vitamins A and B, and proteins of 
good quality, the animals grew and remained in a fairly good 
state of nutrition for varying periods, depending on the season 
of the year. After about forty days on this diet we noted 
that the animals did not have full control of their hind legs. 
They had a peculiar gait due to a partial loss of control of 
the posterior extremities. The gait was tottering and the 
hind quarters wavered slightly from side to side. When 
the animal started to move off rapidly it hopped, usually 
favoring one hind leg. This diet always produces a florid 
condition of rickets, and calcification of the cartilage is 
wanting. The picture of rickets is much more exaggerated 
than that seen in human beings. If this diet is fed with 2 
per cent of calcium carbonate instead of 3 per cent, the histo- 
pathologic picture is identical with that seen in the rickets 
of children. 

Diet 3,143 produces the following changes in the bones of 
rats: the bones are very soft; the ends are much enlarged; 


186 LECTURES ON NUTRITION 


the enlargement of the ends of the long bones is due to an 
increased depth and probably also to an enormous met- 
aphysis; the cartilage in every animal is entirely free from 
lime salt deposits; some irregular lime salt deposition is 
sometimes present in the metaphysis of certain animals; 
the cortex is thin and composed of bone with scanty central 
cores of calcified material having broad osteoid borders. 
The breadth of the osteoid borders is extreme in these ani- 
mals. Few osteoblasts, or at least cells which could be identi- 
fied as such, could be found lining the osteoid trabecule. 
The evidences of resorption of the calcified portions of the 
trabecule were slight. 

Animals which show this wide metaphysis free from cal- 
cification are ready to be used for determining the presence 
or absence of vitamin D in foodstuffs. They must at this 
point be kept in individual cages in order that the food 
consumed can be recorded. McCollum, Simmonds, Shipley, 
and Park (1922) demonstrated that animals in this condition 
will produce a positive ‘“‘line test’”’ if allowed to starve. How- 
land and Kramer (1922) showed this calcification to be 
accompanied by changes in the calcium and phosphorus in 
the serum. They found the serum: of rats on diet 3,143 to 
contain about 10.5 mg. of calcium and about 3 mg. of phos- 
phorus per 100 c.c. of serum. During starvation the phos- 
phorus increases to three or four times this amount. This 
produces the healing seen in starvation of these rachitic rats. 
In testing animals for the antirachitic properties of food- 
stuffs it has been our custom to examine the distal end of 
the left femur and the proximal end of the left tibia for re- 


OUR PRESENT KNOWLEDGE OF VITAMINS 187 


formation of the provisional zone of calcification. The bones 
are split in two with a sharp scalpel and one-half of each 
is fixed in 10 per cent formaldehyde, decalcified in Miiller’s 
fluid and embedded in parloidion. The other half of each 
bone is immersed in 1 per cent silver nitrate and exposed 
to the sunlight or to the light of a Mazda lamp and studied 
through a binocular microscope. 

Rats which gave a positive “‘line test”’ differ from the con- 
trols in having a broad linear deposit of calcium salts on the 
metaphyseal side of the epiphyseal cartilage. The band, 
which may not be complete, is separated from the shaft 
of the bone by the depth of the metaphysis and from the 
nucleus of ossification of the epiphysis by the depth of the 
epiphyseal cartilage. It can be seen on a freshly cut surface 
of an untreated bone as a yellow line which marks the epi- 
physeal border of the metaphysis. The deposit is blackened 
by i per cent silver nitrate in gross specimens. It appears 
like a cross section of a black honey-comb when it is examined 
with a binocular microscope. The metaphysis of these bones 
usually appears to be congested. The calcium salt deposit 
is in the proliferative zone of the cartilage. It may extend 
completely across the bone or may be interrupted or frag- 
mentary according to the activity of the calcium-depositing 
substance. It is stained brown in permanent sections by 
silver nitrate, or an intense blue by hematoxylin. Rats fed 2 
per cent of cod-liver oil for five days give a positive line test. 

Although we presented evidence (1922) which was all but 
conclusive that there is’in cod-liver oil and butter fat a cal- 
cium-depositing substance distinct from vitamin A, it could 


188 LECTURES ON NUTRITION 


not be looked on as finally proved. Our experience with diets 
very low in calcium, but containing cod-liver oil or butter 
fat, demonstrated that 1 per cent of cod-liver oil was very 
superior to 20 per cent of butter fat with these diets. The 
possibility remained that butter fat and cod-liver oil con- 
tained one and the same substance (vitamin A), but that 
cod-liver oil contained much more of this. Zilva and Miura 
(1921) stated their belief that cod-liver oil was 250 times as 
potent as butter fat as a source of vitamin A. Our experi- 
ence had convinced us that existing methods were incapable 
of differentiating beyond doubt between vitamin A and a 
special calcium-depositing substance, should this exist. 
We formulated a plan which involved a comparison of a 
selective list of fats in respect to three kinds of effects in 
nutrition: (1) We tested cod-liver oil, shark-liver oil, butter 
fat, and several vegetable oils for their potency in causing 
the cure of xerophthalmia due to the lack of vitamin A; 
(2) we made comparative tests of the same fats to determine 
their value in promoting growth in young rats which were 
restricted to a diet so low in calcium that satisfactory growth 
was not possible without the provision of some substance 
which would make for greater efficiency in the utilization 
of calcium than that which could be effected in its absence; 
and (3) we further studied these same fats by means of our 
“line test” to discover their relative values for inducing 
the deposition of the line of calcium salts in rachitic bones. 
With the data secured from these three distinct types of test 
we were able to interpret accurately the results of much of 
the confusing experimental data in the literature. 


OUR PRESENT KNOWLEDGE OF VITAMINS 189 


Space will not permit of a detailed exposition of these ex- 
periments. It must suffice to refer to our original papers 
and to a somewhat detailed account which will be found in 
the third edition of my book, “The Newer Knowledge of 
Nutrition.” 

Two to 3 per cent of fish-liver oils or of butter fat were 
found to effect a prompt cure of incipient xerophthalmia 
under the conditions of our test. On the other hand, 8 to 20 
per cent of various vegetable oils failed to cure the eye con- 
dition after it had once developed. Special mention should 
be made of the fact that 15 per cent of cocoanut oil did not 
cure or prevent xerophthalmia. Cocoanut ‘oil was the one 
vegetable oil which we studied which exerted a slight but 
demonstrable effect in causing the healing of the rickets lesion. 

Hopkins (1920) was the first to point out that oxidation 
destroys vitamin A. He showed that if oxygen was allowed 
to pass through heated butter fat the vitamin A was readily 
destroyed. With this treatment butter fat lost its power 
to induce growth or to cure ophthalmia of dietary origin. 
Mellanby (1921) attempted to make use of this means of 
destroying vitamin A in order to determine whether there 
is a distinct antirachitic substance. He found butter fat 
of little value for protecting against rickets after it had been 
oxidized, whereas cod-liver oil after the same treatment, 
that is, heated to 120° C. for four hours while oxygen was 
passed through it, still protected his animals against rickets. 
He stated, “‘If it should happen that four hours’ heating and 
oxidation at 120° C. also leaves a large amount of fat-soluble 
A in cod-liver oil, it will go a long way, especially when con- 


190 LECTURES ON NUTRITION 


sidered together with the butter results, to clinch completely 
the identity of fat-soluble A and the antirachitic vitamin.” 
Mellanby used no method of testing for vitamin A as distinct 
from the calcium-depositing substance, since he did not make 
use of the ophthalmia test. 

We found that cod-liver oil treated with a stream of air 
bubbles at a temperature of boiling water for twelve or 
twenty hours no longer contained sufficient vitamin A to 
relieve rats from xerophthalmia when administered to the 
extent of 2 per cent of the diet. Untreated cod-liver oil . 
under these conditions invariably causes complete recovery 
within five days. ‘Two per cent of fresh butter fat under 
exactly comparable experimental conditions effects the dis- 
appearance of ophthalmia within five to ten days. 

We found 2 per cent of oxidized cod-liver oil very effective 
in causing the healing of the lesion of rickets, notwithstanding 
the fact that its potency for the cure of ophthalmia had been 
lost. Since it is possible to destroy by oxidation the vitamin 
A content of cod-liver oil and still preserve its antirachitic 
potency, we interpreted our experimental data as showing 
that the antirachitic substance is distinct from vitamin A. 

Cod-liver oil is said by Darbey to have been used by phys- 
icians at the Manchester Infirmary in 1789, but Guy (1924), 
who has made a study of the whole literature of cod-liver oil, 
states that its recognition as a curative agent cannot be traced 
to any person, time, or place. The merits of cod-liver oil as 
a preventive or curative agent in rickets is now known to be 
due to its high content of the substance to which the name 
vitamin D has been assigned. No adequate explanation was 


OUR PRESENT KNOWLEDGE OF VITAMINS 191 


available for the absence of rickets in tropical regions, but 
the last few years have seen the accumulation of convincing 
evidence that sunlight, like cod-liver oil, exerts a protective 
influence on the metabolism. 

Zucker, Johnson, and Barnett (1922) report the production 
of rickets in rats on a diet containing an excess of base over 
acid. When they substituted calcium chlorid for calcium 
lactate in equivalent amounts they increased the acidity 
of the diet. The bones of rats on this diet were nearly normal 
or showed mild rickets. The addition of 2 per cent of am- 
monium chlorid, they stated, prevented the development 
of rickets. They believe that a diet which, from the point 
of view of balance between calcium and phosphorus, should 
not lead to rickets, may do so when there is brought about 
a situation to which the acidity of the intestinal tract is 
lessened. : 

Pappenheimer and co-workers (1922, 1923) have discussed 
the effects of varying the inorganic constituents of a rickets- 
producing diet. Inorganic salts other than calcium or phos- 
phate seemed to be without influence on the development 
or prevention of rachitic lesions. 

Bosanyi (1924) has reported remarkable experiments in 
which he showed the subcutaneous administration of aqueous 
extracts of normal bone-marrow causes the healing of ra- 
chitic bones in the rat. The phosphorus content of the ex- 
tracts was very small, so the effects could not be attributed to 
the element. He believes that the failure of the initial de- 
position of calcium in rickets is due to the stoppage of the 
biological functions of the bone-marrow through which nor- 


192 LECTURES ON NUTRITION 


mally a permanent deposition of calcium is insured. Normal 
functioning bone-marrow produces a substance which is 
transmitted to the tissues requiring to be calcified which 
renders them capable of calcification. ‘This biologic factor 
in an active state can be extracted with water from the 
healthy marrow, but this is not identical with the enzyme 
present in the watery extract. His experiments demonstrate 
that this factor is not produced in the marrow of rachitic rats. 

The fat melted from bone-marrow had a slight protection 
if melted from the red bone-marrow, but no effect if from yel- 
low marrow. ‘The marrow wholly defatted protected animals 
against rickets. When filtered through collodion, aqueous 
extracts of marrow had no antirachitic effect. Dialyzed ex- 
tract had no effect, whereas the gelatinous remainder not 
dialysable had a markedly antirachitic effect. Heating to 
70° C. did not affect the properties of the extracts, but higher 
temperatures destroyed it. Extracts were made of liver, 
spleen, thymus, thyroid, and pancreas, of which the spleen 
extracts only were found to be antirachitic. Extracts of the 
spleen of a rachitic animal were inactive. Extracts of bone- 
marrow of rachitic rats which had starved from four to five 
days and showed healing were not antirachitic. The substance 
is present only in normal bone-marrow. Bosanyi employed 
the “‘line test” procedure. 

Bethke, Steenbock, and Nelson (1923, 1924) raised the 
question as to whether or not some of the beneficial results 
recorded in the literature on feeding of calcium salts may 
not have been due to mass action of the calcium counteracting 
the effects of vitamin deficiency. They found where no fat- 


OUR PRESENT KNOWLEDGE OF VITAMINS 193 


soluble vitamins were added the calcium of the blood was 
constantly depressed, but apparently the greatest depression 
took place when the most phosphate was added. 

Zucker, Pappenheimer, and Barnett (1921) stated that the 
antirachitic principle is found in the ether-soluble, unsaponi- 
fiable fraction of cod-liver oil after alkali hydrolysis. This 
we have ourselves observed. The fatty acids of cod-liver 
oil are entirely inactive in curing rickets. Zucker (1922) 
reported that a good yield of a crude product of the anti- 
rachitic vitamin could be obtained by extracting cod-liver 
oil with 95 per cent alcohol. The mixture of fatty acids in 
a small amount of oil and other substances was saponified 
with NaOH. The calcium soaps were then precipitated and 
ultimately extracted with acetone. The acetone extract is 
exceedingly potent. 

Howland and Marriott (1918) discussed the theories as 
to the causation of infantile tetany. Tetany has been re- 
ferred to: (a) Dysfunction of the parathyroid gland; (6) the 
character of the food; (c) intoxication by calcium; (d) intoxi- 
cation by guanidine, or methylguanidine; and (e) lack of 
calcium. ‘They stated that the evidence in 1918 failed to 
support most of these theories, although there had been in 
the literature for a hundred years satisfactory descriptions 
of infantile tetany. They pointed out that normal calcium 
of the blood is 10 to 11 mg. per 100 c.c. of serum. In some 
cases of rickets they found a lowering of the calcium to 8 mg., 
although in some cases of rickets the calcium remained normal. 
In tetany during the active symptoms the calcium content 


of the serum was invariably reduced and may fall as low as 
13 


194 LECTURES ON NUTRITION 


3.5 mg. In convulsive disorders other than tetany they found 
no reduction of the calcium of the serum. They found cal- 
cium chlorid given by mouth caused prompt relief of spas- 
modic symptoms. Howland and Kramer (1921) reported 
studies on the determination of the inorganic phosphorus of 
the serum. Their results represent the orthophosphoric 
acid content of the serum, the only form of phosphorus which 
can react with calcium to form tertiary calcium phosphate. 
They believe there can be no doubt that the inorganic phos- 
phorus represents a definite chemical entity, since it is present 
in nearly constant amounts in the same individual as well as 
in normal individuals of the same age. Their studies show that 
in rickets there is a marked decrease in inorganic phosphorus 
in the serum. To this deficiency they ascribed the failure 
of calcium deposition. Non-rachitic infants and young chil- 
dren have between 10 and 11 mg. of calcium and about 
5 mg. of inorganic phosphorus per 100 gm. of serum. 

We found (1922) that a pathologic condition correspond- 
ing in all fundamental respects to rickets in human beings 
can be produced by diet in two ways: by diminishing the 
phosphorus and supplying an optimal or an excess of cal- 
cium, and by reducing the calcium and maintaining the phos- 
phorus at a concentration somewhat near the optimum. As 
a result of these experiments we were led to believe that 
there are two kinds of rickets, one being characterized by a 
normal or nearly normal blood calcium and a low blood phos- 
phorus (low phosphorus rickets; calcium to phosphorus ratio 
large); the other being normal or nearly normal blood phos- 
phorus with a low blood calcium (low calcium rickets; low 


OUR PRESENT KNOWLEDGE OF VITAMINS 195 


calcium to phosphorus ratio). The investigations of How- 
land and Kramer (1921) and of Kramer, Tisdall, and How- 
land (1921) on the calcium and phosphorus content of the 
blood serum in rickets and tetany supported this view. 
These observers found that in children suffering from rickets 
alone the phosphorus of the blood serum is low and the 
calcium about normal. In children suffering from tetany 
complicating rickets, on the other hand, the calcium is low, 
but the phosphorus not far from normal. They believe that 
tetany is an expression of the nervous tissues of an insufh- 
ciency of the calcium ion, and that rickets is essentially an 
expression on the part of the skeleton of disturbed relations 
between the calcium and phosphate ions of the body fluids. 
They stated that tetany is frequently associated with rickets 
because rickets is a disease in which the calcium ion in the 
body tissues and fluids is subject to variation. Tetany occurs 
independently of rickets just as rickets occurs independently 
of tetany. Since tetany may occur with a low phosphorus 
form of rickets it does not serve to mark off one form of rickets 
from the other. Tetany is essentially associated with a low 
calcium form of rickets and, for all practical purposes, the 
low calcium form of rickets is the rickets of tetany. 
Howland and Kramer (1923, 1924) found that in uncom- 
plicated cases of rickets the concentration of calcium in the 
serum is normal or nearly so, whereas the phosphorus con- 
centration is regularly low, so that the product of the two 
concentrations is at or below 30. In tetany the product is 
also low on account of the striking reduction in the calcium. 
In all of these cases rickets was present. After cod-liver oil 


196 LECTURES ON NUTRITION 


or ultraviolet-ray therapy the phosphorus rises so greatly 
as to make the product two or three times what it was before. 
The graph taken from their paper was constructed from figures 
for the concentration of calcium and inorganic phosphorus 
both of children and of rats. This illustrates clearly the signifi- 
cance of the products of these concentrates. On the abscissa 
are marked the concentrations of phosphorus; the oblique 
lines represent the concentrations of calcium and the prod- 
ucts may be read off from the ordinates. The cases of active 
rickets occurring in children are represented by a circle, and 
those in rats by a triangle. Healing is indicated by a dark 
circle or by a dark triangle. It will be noted that below the 
horizontal line corresponding to the figure 30 on the ordinate 
all the triangles and circles are light. Above 40 they are all 
dark. Between 30 and 40 only one is dark. ‘This graph is 
to be interpreted that when the product of calcium and 
phosphorus figures is below 30, rickets is to be expected; 
between 30 and 40 it is impossible. When the product is 
above 40 either healing is taking place or rickets is entirely 
absent. 

Petersen (1924) reported an experimental study of ununited 
fractures with special reference to the inorganic bone-form- 
ing elements in the blood serum. He applied the principles 
elaborated by Howland and Kramer, and by McCollum, 
Simmonds, Shipley, and Park in their studies on experi- 
mental rickets in the rat. He found it possible by dietetic 
management to lower the phosphorus content of the blood 
of dogs to a point where the product of calcium and phos- 
phorus figures was less than thirty. He concluded from his 


OUR PRESENT KNOWLEDGE OF VITAMINS 197 


study that in the healing of fractures a definite relationship 
exists between the concentration of the inorganic bone- 
forming elements in the serum and the rate of repair. If 
the phosphorus and the product of the calcium times and 
phosphorus figures are again raised to their normal level, 
the fractured bones will unite. This condition he was able 
to establish by dietetic management. 

The Effect of Light in the Prevention and Cure of Rickets.— 
There has long been a belief among clinicians that sunlight, 
calcium-rich food, and cod-liver oil are of value in the treat- 
ment of disease, especially rickets and tuberculosis. Palm 
(1890), as a result of topographic study of the incidence 
of rickets, believed that sunlight should be looked on as a 
therapeutic agent. Buchholz (1904) successfully treated 
rickets with artificial light. Neumann (1909) observed that 
rickets and tetany did not occur at high altitudes in Swit- 
zerland, but was found at lower altitudes where the sunlight 
was less intense. In 1912 Raczynski wrote: “It is the sun 
which plays the principal réle in the etiology of rickets.” 
He gave the first proof of the favorable influence of light on 
mineral metabolism by an experiment on puppies. One 
puppy was reared in the dark, the other in sunlight, and both 
were suckled by the mother. At the end of six weeks he found 
the calcium and phosphorus in the body of the puppy reared 
in-darkness was very much less than that of the one reared 
in sunlight. Commenting on his studies he says, “It is pos- 
sible to assume that the lack of action of sunlight, by influen- 
cing in so unfavorable a manner the assimilation of calclum 
oxid in the young organsim, is one of the causes of rickets.”’ 


198 LECTURES ON NUTRITION 


Huldschinsky (1919) reported that the ultraviolet ray ex- 
erted a curative action on rickets. Winkler (1918), Riedel 
(1920), Erlacher (1921), Mangert (1921), Hess and Unger 
(1921) extended these observations and established the 
therapeutic value of light. Sachs (1920, 1921) and Huld- 
schinsky (1920) reported the cure of tetany with light treat- 
ment. Shipley, Park, McCollum, and Simmonds (1921) and 
Hess (1921) observed the prevention of rickets in rats by 
exposure to sunlight. 

Hume (1922) found that irradiation with a quartz mercury 
vapor lamp prolonged the growth of rats on a diet free or 
almost free from vitamin A. She concluded that there is 
an interaction of light and vitamin A in the growth of rats, 
but no photosynthesis of the vitamin. Goldblatt and Soames 
(1923) irradiated rats for four weeks which were declining 
in weight due to vitamin A deficiency. Livers of these rats 
were fed to other rats on a vitamin A free diet and the latter 
responded with growth. ‘They raised the question as to 
whether or not vitamin A was synthesized. Irradiated rats 
on a diet free from vitamin A do not continue to grow, al- 
though they do grow better for a time than rats not irradi- 
ated. However, they finally lose weight and die like those not 
irradiated. Since this is true, why did the livers of rats which 
were declining in weight, which were then irradiated, make 
other rats grow? They do not interpret their results as 
indicating a synthesis of vitamin A, but are unable to explain 
them. 

Hume and Smith (1923) reported that when rats are fed a 
diet deficient in fat-soluble vitamins, but kept in glass jars 


OUR PRESENT KNOWLEDGE OF VITAMINS 199 


which have been exposed to the mercury vapor lamp for ten 
minutes every second day, they grew better than the controls 
not so treated. Rats exposed to air containing ozone, drawn 
over the quartz lamp and passed through 3 meters of glass 
tubing, showed poorer growth than the control animals. 
The same rats subsequently treated in glass jars filled with 
irradiated air gave some growth response. The growth of 
rats placed in irradiated jars from which the irradiated air 
had been displaced is not prolonged. 

Webster and Hill (1923) were unable to confirm the ob- 
servations of Hume and Smith. They also tried without 
stimulating effect: ozone, dilute NOs, gas, air in glass jars 
which had been exposed to x-ray tubes and which was pre- 
sumably ionized, and cigarette smoke. They found that 
exposure for thirty minutes daily to air led from the enclos- 
ure of the mercury vapor lamp failed to influence rickets in 
young rats on a deficient diet. 

Steenbock and Black (1924) report the surprising observa- 
tion that rat rations irradiated by the mercury vapor quartz 
lamp can be activated so as to make them growth promoting 
and bone calcifying to the same degree as when rats are 
irradiated directly. The activation takes place when the 
ration is irradiated in an open dish or in a stoppered Pyrex 
or quartz flask filled with air or carbon dioxid, but not in 
a brown bottle. The activation is not destroyed by subjecting 
the ration to a vacuum, heating it for forty-five minutes at 
96° C., or letting it stand for twenty-four hours at room tem- 
perature. They confirm the observation of Goldblatt and 
Soames (1923) in finding that liver taken from irradiated rats 


200 LECTURES ON NUTRITION 


is growth promoting, whereas the liver from non-irradiated 
rats is inactive. The same was found true of lung and muscle 
tissue. Inactive muscle, after removal from the body, ex- 
posed to the radiations of the lamp, was found to have become 
activated, being both growth promoting and bone calcifying. 
Liver treated the same way also promoted bone calcification. 
The activity of liver taken from irradiated rats was not des- 
troyed by drying at 96° C. for twenty-four hours and then 
keeping it in a stoppered bottle for two months. 

Hess (1924) states that attempts to prevent rickets in 
rats by means of fluids which contain radium failed. Water 
to which radium bromid had been added was fed daily, but 
without effect. This was likewise true with regard to lin- 
seed oil to which minimal amounts of radium had been 
added. Subcutaneous injections in water were also with- 
out effect. He found that cottonseed oil when rayed for 
an hour with the mercury quartz lamp at a distance of 1 
foot acquired antirachitic potency. Linseed oil was also 
made antirachitic by ultraviolet rays. Mineral oil irradi- 
ated in the same manner gave a negative result. 

Hess and Weinstock (1924) observe that “while it is im- 
possible to state definitely that the substance which is formed 
is the same as that which is responsible for the remarkable 
curative value of cod-liver oil, it has been found by means of 
chemical examinations that cod-liver oil can be separated | 
into two distinct portions, one which is of value and one of 
no value in curing rickets.'. . . Vegetable oils which have 
been exposed to the ultraviolet rays can likewise and by the 
same chemical means be separated into a portion which is 


OUR PRESENT KNOWLEDGE OF VITAMINS 201 


of no value, and another which acts as a specific in warding 
off or curing rickets. It would, therefore, seem that a sub- 
stance has been formed in the vegetable oils by the action 
of the ultraviolet rays similar to that which is naturally 
present in the liver of the cod.” 


VITAMINS AND REPRODUCTION 


Evans and Bishop (1922) have reported studies on the 
relation between nutrition and fertility. They point out 
that animals confined to a certain ration are usually sterile, 
but not invariably so. Ovulation is unrelated to the repro- 
ductive function on this basic diet, since the ovulation rate 
is normal, but there is failure of the females to reproduce. 
The animals conceive and the placente are implanted. In 
other words, early steps in reproduction are not interfered 
with, but about the fourteenth day after a positive mating 
the majority of the females give what Evans and Bishop 
designate as the “placental sign.”’ This consists of a slight 
leakage of blood from the placenta. The vaginal smear con- 
tains blood cells due to a leakage of the placenta. Necrop- 
sies performed at this time show that the sign has been 
given by the placentz, which are very abnormal. In some 
instances the fetuses have been completely absorbed. They 
pointed out that when certain natural foods, for example, 
lettuce, are given to a female, even after one or more resorp- 
tions have taken place, she will produce normal young. 
In summarizing their extensive studies on the production of 
sterility with nutritional regimens they state that the basal 
ration, which was composed of casein, lard, and cornstarch, 


202 LECTURES ON NUTRITION 


with salts, supplemented with yeast as a source of the vita- 
min A, failed to give normal fertility in a large number of 
female rats. ‘They have demonstrated that this sterility may 
be prevented or cured after its appearance in any individual 
case by the addition of certain natural foodstuffs to the 
basal ration. They found that lettuce, meat, whole wheat, 
wheat germ, rolled oats, dry alfalfa, and large quantities of 
milk fat will relieve this condition. Wheat germ oil is said 
to be especially effective even in very small doses. Whole 
milk, fresh or dried, cod-liver oil, orange juice, and yeast 
failed to act as curative agents when added to the basal diet. 
The unknown fertility-conferring factor which they have 
tentatively called “X,’” shown to be present in the above 
list of foods, cannot be identified with the known vitamins 
A, B, C, or D. Sure (1924) has confirmed the observations 
of Evans and Bishop relative to the potency of the ethereal 
extract of yellow corn, wheat embryo, and hemp seed in 
preventing sterility in the rat. He also observed fertility 
when commercial cottonseed oil and commercial olive oil 
were introduced to the extent of 5 per cent into the sterility 
diet, but not with commercial cocoanut, linseed, or sesame oil. 


CONCLUSION 


This, then, is the story of our present knowledge of vitamins , 
and the réle they play in influencing metabolism. Of neces- 
sity the account has been shorn of all details. The field of 
vitamin research, both on the chemical and pathologic side, 
is still a fertile one, and within recent years has attracted 
an ever-increasing number of investigators. Some of the 


OUR PRESENT KNOWLEDGE OF VITAMINS 203 


work recorded is probably open to criticism on the basis of 
faulty dietetic management of the animals, but consider- 


able time will be necessary in order to separate fact from 


error. It is still too early to attempt any general discussion 


of this kind, or a critical examination, especially of the more 


recently recorded observations. 


An Pwn eS 


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a1 4 Barly 

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aan 


v 


THE RELATIONS BETWEEN FERTILITY AND 
NUTRITION* 


HERBERT McLEAN EVANS 


As is well known to most members of this audience, the 
science of nutrition, which may be said to have received its 
classical formulation by the lineage of investigators repre- 
sented by Lavosier, Liebig, von Voit, Rubner, and many 
others, has undergone a striking enlargement in recent years 
by the discovery of the nutritive need of the body for very 
minute quantities of several kinds of chemically unknown 
substances, the vitamins. A summary of our knowledge in 
this field by one who has himself played a significant part 
in delineating it (Professor McCollum) has already con- 
stituted one of the lectures in this series, so that I can the 
better dispense with further elaboration here. 

The discovery of the indispensability of these substances 
may be said to have come especially through efforts to rear 
animals on so-called pure or synthetic foods. The work was 
undoubtedly facilitated by the use of a hardy small mammal, 
sufficiently indiscriminate in its taste to reject little or noth- 


* Part of this address is substantially identical with a report made on 
fat-soluble vitamin E to the National Academy of Sciences at their annual 
meeting, Washington, D. C., April 27, 1925 in conjunction with my asso- 
ciate, Dr. George O. Burr. It should, therefore, be borne in mind that Dr. 
Burr shares in every way in the elucidation of the latter problem. 

14 209 


210 LECTURES ON NUTRITION 


ing edible, the rat. The occurrence of actual disaster, that 
is, death, when either of the two major vitamins, fat-soluble 
. A and water-soluble B, are absent, made the detection of 
these two substances inevitable sooner or later. On the 
other hand, the experimental production of two grave, well- 
characterized diseases, scurvy and rickets, the former in guinea- 
pigs, has led with no less security to the recognition of C and 
D. I shall endeavor to present here in some detail the evi- 
dence which now establishes the existence of the fifth mem- 
ber of the vitamin class, the substance provisionally char- 
acterized by the symbol X, but for which one may use the 
serial designation E. Animals can suffer serious impair- 
ment, especially where it concerns some of the functions of 
the more hidden organs, without that impairment showing 
itself openly. It would consequently not seem too hazardous 
a conjecture for us to expect that refinement in our method 
of measuring the function of organs will lead to the dis- 
covery that the particular chemical work involved in the 
activities of some of the organs and tissues depends on still 
other specific foods. Indeed, one finds that twenty years 
ago this hazard had already been taken by Professor Gow- 
land Hopkins, to whom in part we owe the opening of this 
field of research when he said, ‘“The animal body is adjusted 
to live either upon plant tissues or other animals, and these 
contain countless substances other than the proteins, carbo- 
hydrates, and fats. Physiologic evolution, I believe, has 
made some of these well nigh as essential as are the basal 
constituents of diets.” 


RELATIONS BETWEEN FERTILITY AND NUTRITION 211 


PHYSIOLOGY OF REPRODUCTION OF RATS 

A number of years ago we attempted in the Anatomical 
Laboratory of the University of California a very careful 
analysis of the steps in the physiology of reproduction in 
these small rodents in the hope of utilizing this material for 
experimental procedures in the field of mammalian embry- 
ology. Enough was learned, I feel, to constitute a very 
substantial advance on the previous indefiniteness of our 
knowledge in this interesting realm. As I hope to portray 
presently, it was possible for us to discover in the living 
animal the approach of cestrus and correct time of insemina- 
tion, the time at which the final ripening or maturation 
changes occur (involving the formation of the first polar 
body) and the time at which follicular rupture or ovulation 
actually occurs and the tubal journey begins; furthermore, 
if mating was permitted, the detection of the state called 
pseudopregnancy and, finally, on the thirteenth to the six- 
teenth day, the definite detection of pregnancy through the 
discovery of what we may call the implantation sign. We 
had in our hands then, probably for the first time, an ade- 
quate method for the detection of aberrations in the mys- 
terious processes involved in the physiology of reproduction. 
As a matter of fact, when we turned our attention to the 
possible dependence of reproduction on nutrition we were 
promptly struck by a host of new facts. 

Relation to Nutrition.—Nutritive regimens adequate for 
livelihood, indeed, for approximately normal growth and for 
the appearance of health in animals, are still inadequate for 
the maintenance, for instance, of the normal rhythm of 


212 LECTURES ON NUTRITION 


ovulation. And when we turned to the study of reproduc- 
tion we were astonished to find it fail in a large proportion 
of animals reared on pure foods and an abundance of the 
hitherto established vitamins, particularly A and B. Small 
amounts of some natural foods cured the disorder or sterility 
provoked by these regimens, a disorder which was hence a 
true dietary deficiency disease. Furthermore, the beneficial 
properties of these curative foods could be extracted from 
them and, in turn, other satisfactory extracts made from these 
extracts which are at this point very concentrated and need 
to be fed only in mere traces. We have hence, I believe, ade- 
quate evidence before us for declaring the existence of a 
fifth class or group of the vitamin substances: the sub- 
stance fat-soluble E. 

Before turning specifically to the data which bear on the 
discovery of the new vitamin, on its occurrence in various 
foods, on its physical and chemical characteristics, and the 
present stage of its isolation, it would seem best to prepare 
the ground, as it were, by reverting to our general studies 
on the reproductive system and to describe also briefly some 
of the other relations between fertility and nutrition, a field 
of which the vitamin studies constitute but a part and one 
which holds promise of important rewards to the investi- 
gator who will explore it with the criteria now available for 
advances in these newer aspects of mammalian embryology. 
That there are many relations between fertility and nutri- 
tion (some of them at present unconjectured) is, I believe, a 
fact. 

Vaginal Changes During the CEsirual Cycle.—The precision 


RELATIONS BETWEEN FERTILITY AND NUTRITION 213 


with which one may detect the periodicity in ovarian func- 
tion in some laboratory animals is truly remarkable. Many 
years ago Morau, Lataste, Retterer, and K6nigstein described 
changes in the character of the epithelium of the genital 
tract and especially the vaginal canal in several common 
types of mammals. They made out definite changes, for 
instance, associated with the phenomenon of heat, or rut, 
but in 1917 Stockard and Papanicolaou showed that micro- 
scopic examination of the cell detritus and exudate in the 
vaginal canal (from samples which can be obtained with 
maximal celerity and ease in the living animal) enable us 
to determine the exact time of cestrus and ovulation and 
to segment the cestrual cycle into definite periods or steps. 
When we examined the rat in this way we had no difficulty 
in making out the existence of similar steps or periods in 
the cestrual cycle, which recurs with striking regularity every 
four or five days. In these forms the mucous membrane of 
the vaginal canal exhibits a period of growth, differentiation, 
and desquamation, the various steps of which take place in 
so orderly a sequence as to furnish us more information 
about the approach and incidence of ovulation than is fur- 
nished, for example, by the monthly appearance of a san- 
guineous discharge in the human being. In fact, cyclic 
changes affect the entire genital canal from the oviduct 
to the vaginal orifice. The ovary presides over or has some 
necessary connection with these changes, for on its ablation 
the changes disappear never to recur save on successful 
ovarian transplantation, when they may be again just as 
regular. We are even now in ignorance as to the exact time 


214 LECTURES ON NUTRITION 


relation between human menstruation and ovulation. The 
relation of cestrus to ovulation has been somewhat clearer, 
for the two events have always been recognized as close to- 
gether in time. Our newer studies, however, inform us as. 
to the exact time relations of these two phenomena, ovula- 
tion occurring at a precise time interval after cestrus. By 
killing animals at the various steps in what is an orderly 
progress of changes in the cellular character of the vaginal 
smear, it is possible to relate these changes with certainty 
to the time of ovulation. Immature animals, pregnant ani- 
mals, or those from which the ovaries have been ablated do 
not, as has already been indicated, ever show these changes 
in the character of the vaginal smear, but, when ovarian 
follicles begin to grow and rupture, the cell changes invari- 
ably occur. Though we know many details about these 
changes, the essential facts are briefly as follows: 

Irregularly shaped, small, nucleated epithelial cells mixed 
with leukocytes constitute the picture found in the resting 
stage. Preparatory to cestrus and ovulation there is a sharp 
pause or halt in the immigration of leukocytes which usually 
creep in in considerable numbers from the subjacent capil- 
laries through the epithelial cells of the vaginal mucosa 
into the lumen. Leukocytes are suddenly no longer en- 
countered in the smear. Epithelial cells alone, a peculiar 
type of them, sometimes in sheets, are now found. Quickly 
succeeding this change come non-nucleated, transparent 
cornified cells, sooner or later in similar sheets. A massive 
production of these cells takes place, so that macroscopically 
a cheesy detritus is found. Eventually leukocytes again 


RELATIONS BETWEEN FERTILITY AND NUTRITION 215 


recur and quickly thereafter are found in enormous num- 
bers. The cornified epithelial cells give way to scanty small 
nucleated ones. This is the cycle of changes. The presence 
of true cornified cells, singly or in sheets, is a reliable cri- 
terion for impending maturation of ova and consequent 
rupture of Graafian follicles in the ovary. 

In the case of a large colony of animals, three persons 
working as a so-called diagnostic group can thus remove 
them from their cages, carry to the examining table and 
register on individual record cards the cell types found in 
every individual daily. The individual smears are quickly 
tapped into a drop of salt solution and the cell types floating 
there diagnosed almost at a glance with the low power of 
the microscope, the diaphragm being turned well down. In 
this way the cestrual cycles have been followed in the cases 
of every animal studied by us. 

Effect of Vitamins A and B on Ovulation and CGstrus.— 
When, a number of years ago, animals were reared and 
maintained on a classic pure dietary mixture consisting 
essentially of casein, cornstarch, lard, butter, and salts, to 
which an appropriate separate dose of dried yeast was added 
daily for vitamin B, it was soon noted that while all other 
phenomena were apparently normal, the animals showed late 
maturity and infrequent ovulations. When the yeast dosage 
given these animals was greatly increased, for instance, from 
a daily quantity of 100 mg. to one of 400, 500, or 600 mg., 
the ovulation rate became normal, that is, ovulation re- 
curred every four or five days. It was apparent, then, that 
we had a new and more sensitive test for physiologic well- 


216 LECTURES ON NUTRITION 


being than that furnished by either growth, glossy coats, 
bodily activity, or the other easily detectable signs. 

When we came to study the situation with reference to 
vitamin A, equally interesting discoveries were made. As 
is well known to you, food impoverishment in this vitamin 
has for its sequel, sooner or later, trouble with the secre- 
tion of the lacrimal gland into the conjunctival sac and 
with the nutrition of the cornea, so that the occurrence of 
the so-called xerophthalmia is practically pathognomonic 
for deficiency of vitamin A. Nevertheless, it has long been 
recognized that xerophthalmia can be prevented, or at any 
rate greatly delayed, by satisfactory care of the eye, that it 
may be present in one eye alone, or that, indeed, it need not 
occur in animals when we are certain that death is due to 
what I have briefly designated as “A disease.” Slowing 
and cessation of growth, and ultimate decline in weight also 
occur in these conditions, but they are not solely charac- 
teristic, of course, of this disorder. It was therefore highly 
interesting that in our attempt to study the sex cycles of 
animals impoverished in A, a new and characteristic sign of 
lack of this vitamin was quickly disclosed. We found that 
the tendency on the part of the vaginal mucosa to form 
cornified epithelial cells was no longer limited, as is normally 
the case, to the time of growth, maturation and rupture of 
the Graafian follicles, but that in A disease the desquamation 
of cornified cells was continuous and hence obscured all 
ovarian cycles that may also have been present. Further- 
more, the new sign is not mediated through the ovary; that 
is, A impoverishment does not create a malfunction, as it 


RELATIONS BETWEEN FERTILITY AND NUTRITION 217 


were, of the ovaries which thus continuously secrete hor- 
mones similar to those in oestrus, for the new sign is given 
in the presence of A disease even if the ovaries are ablated. 
It is not improbable in explanation of the new sign that we 
have to do with a disturbance of water metabolism and, in 
the case of the mucous membrane in question, again with 
xerosis. Studies on this point are in progress. I would only 
emphasize here the fact that the sign has been a valuable 
instrument in dietary research. It is given by no other 
food deficiency known to us save that of vitamin A, and in 
the study of many cases it has not only been found to occur 
with complete regularity, but to occur as early as any other 
sign, for example, decline in growth or ophthalmic disease, 
and in most cases earlier than other signs. Animals may be 
fed mixtures of the most miscellaneous foodstuffs, but seri- 
ous deficiency in vitamin A will not be disguised by other 
inadequacies. By feeding subnormal amounts of vitamin 
A, ophthalmic disease may be prevented, and growth, in 
fact, kept practically normal, whereas deficiency in the sub- 
stance A can still be frequently detected by the continuous 
exhibition of the new sign on the part of otherwise appar- 
ently healthy animals. Raising the level of A immediately 
abolishes the sign. Whether the sign is given in still slighter 
deficiencies in A (by cases, for instance, in which, as Sherman 
has shown, only lactation or longevity is affected) remains 
still to be shown.* 


* Since the foregoing was written Wolbach and Howe have reported on 
the extensive transformation of epithelium in various parts of the body 
into a stratified, squamous keratinizing epithelium, calling attention 
particularly to the upper respiratory tract and to the renal pelvis, the 


218 LECTURES ON NUTRITION 


VITAMIN E 


I shall now turn to the characterization of the sterility 
disease produced by pure foods or other dietaries lacking the 
substance which we have called fat-soluble E. When rats 
are reared on some “synthetic” food mixtures consisting of 
fat, carbohydrate, and protein in separate relatively pure 
form together with an appropriate salt mixture and the 
vitamins A and B,* they grow well and have every appear- 
ance of health. Depending somewhat on the exact char- 
acter and the proportions of the constituents of the food, 
they sooner or later exhibit complete sterility. In many 
instances a transitory period of fertility, variable in length, 
follows the attainment of sexual maturity. This is usually 
the case with the male, but it is also frequently the case 
with the female. We are as yet imperfectly informed as to 
all the factors which may delay or prevent the onset of this 
peculiar form of dietary sterility which, as will be shown 
below, can be so spectacularly cured by small doses of vita- 
urinary bladder, and the seminal vesicles, epididymis, and prostate. The 
salivary glands and pancreas participate in the change. They do not note 
the change in the vaginal epithelium, the only epithelium from which 
samples may be removed with ease and at will in the living animal. 

* Various proportions of casein, lard, butter, and cornstarch have been 
used, the commonest being that after Osborne and Mendel as follows: 
Casein 18, cornstarch 54, lard 15, milk fat 9, salts 4, 0.4 to 0.6 gm. dried 
yeast daily, the vitamin B being secured from daily administration of 0.4 to 
0.6 gm. of whole dried yeast and A from the butter employed. In many in- 
stances, however, milk fat was omitted and the A requirements met with 
various levels of cod-liver oil which varied from a single drop daily to 2 
per cent by weight of the ration. The salt mixture employed was after 
E. V. McCollum and consisted of NaCl 0.173, MgSO, (anhyd.), 0.266, 


NaH2PO, + HO, 0.347, KePOu,, 0.954, CaH4(PO,)2 + H:0, 0.540, citrate 
of iron, 0.118, calcium lactate, 1.300. 


RELATIONS BETWEEN FERTILITY AND NUTRITION 219 


min E. The preliminary fertile period may, in fact, be very 
extensive, and it is this doubtless which has led some investi- 
gators to doubt the existence of a new specific vitamin essen- 
tial for reproduction.* We may possibly with justification 
point to an analogy here with the regulation of inorganic 
metabolism and especially that of calcium and phosphorus 
by means of vitamin D, for it would appear from the re- 
searches of McCollum and others that it is the ratio between 
the inorganic elements in question rather than their absolute 
abundance in the diet which occasions disorder in ossification. 
It would appear that if the proper ratio is maintained animals 
will not develop rickets, but that, on the other hand, small 
amounts of vitamin D will enable animals to overcome very 
unfavorable ratios in the elements in question. In the case 
of young from females reared on a “pure” food ration, the 


* We have found, for instance, that when lard is omitted from the 
diet, 5 per cent butter fat often suffices to confer fertility on the animals 
throughout the early portion, and in some cases the greater portion of 
the life span. With a dietary mixture of casein 18, cornstarch 73, milk-fat 
5, and salts 4 we have had from twenty-six females a total of sixty-six 
litters, totaling 304 young, born during a time when litter-mate sister con- 
trols held on casein 18, cornstarch 63, lard 10, milk-fat 5, and salts 4 pro- 
duced no young. It would appear from these facts that, although the 
actual amount of vitamin E which we know to be present (although low) 
in}milk-fat must be the same in both of these diets, the lard exercises in 
some way a sterility producing effect. The explanation of such phe- 
nomena given by V. E. Nelson, that the higher fat quota alone is respon- 
sible for the sterility, cannot be maintained inasmuch as we have employed 
diets containing 24 per cent by weight of milk-fat with fertility invariably 
resulting. It is perhaps well to note that animals on the first or fertile 
ration, are not invariably fertile, and furthermore that while the major- 
ity are at first fertile, they also sooner or later tend to lose their fertility, 
and in those cases in which sterility supervenes, it is promptly cured 
by the administration of very small doses of vitamin E as hereinafter 
described. 


220 LECTURES ON NUTRITION 


young secured either from the early fertility period or by 
fertility induced by certain food extracts hereinafter described, 
we usually observe complete sterility from the very beginning 
of sexual life. We have abundant evidence that sterility 1s a 
dietary deficiency disease, for it can be cured or prevented by a 
change in dietary regimen, a change involving the addition of 
certain single natural foods high in a new food factor, vitamin 
E,* or the addition of very much smaller amounts of extracts of 
these foods. ‘The sterility disease affects males and females 
differently. 

In the male it eventually leads to destruction of the germ 
cells (eventually the entire seminiferous epithelium), but this 
is not the case with the female, where the ovary and ovulation 
are unimpaired throughout life, but where a highly character- 
istic disturbance occurs in gestation, namely, the death and 
resorption of the developing young. 

“Resorption Gestation.”’—It is necessary to insist on the 
peculiar character of dietary sterility thus produced in the 
female through lack of vitamin E, for it is only by ascer- 
taining the existence of typical “‘resorption gestations” that 
one may be assured that he is dealing with deficiency in the 
specific substance E. Many other dietary deficiencies cause 
sterility in the female, but they all do so by interference 
with other steps in the reproductive mechanism than those 
involved in lack of E, usually by preventing cestrus, ovula- 
tion, fertilization, or implantation, but not by resorption 


* Provisionally designated in previous publications from this laboratory 
as vitamin X. We now designate it E on account of its serial position 
following the alphabetic terminology proposed by McCollum, who named 
the antirachitic factor D. 


RELATIONS BETWEEN FERTILITY AND NUTRITION 221 


after implantation has occurred. In order to establish fe- 
male sterility as due to absence of fat-soluble vitamin E, it 
is necessary to establish with certainty the existence of 
cestrus and ovulation, coition, and implantation. Such in- 
formation is best secured by use of the newer methods of 
studying the vaginal smear, by mating animals at the appro- 
priate time in the cestrual cycle, by subsequent detection of 
the “bouchon vaginale” and residual sperm, of the cessation 
of cycles, and finally, on the fourteenth to sixteenth day of the 
occurrence of erythrocytes in the smear, a positive sign of 
implantation. In gestations where E is low or absent, the 
embryos seem at first normal, but sooner or later, often by 
the eighth day, retardation in development can be demon- 
strated. Evident abnormality, especially monstrosity, does 
not occur. At some time between the twelfth and twentieth 
day fetal death occurs, usually on the twelfth or thirteenth 
day, but for some days thereafter the maternal part of the 
placenta continues to live. There may also be continued gain 
in the mother’s weight until the twentieth or twenty-first 
day. This would appear to speak decisively for peculiar need 
on the part of the developing young for the new vitamin as 
against placental injury as the cause of death. Furthermore, 
it would appear that the maternal placenta is not altered 
structurally sufficiently to justify the conclusion that its 
function had been impaired. Subnormality is seen not only 
in the embryo, but in the fetal parts of the placenta, both in 
yolk sac and allantois, especially in the former; in the yolk 
sac underdevelopment of the entodermal villi and blood 
islands is conspicuous, whereas, in the embryo one may note 


222 LECTURES ON NUTRITION 


impairment in the mesenchyme and its chief derivatives, the 
blood-vessels and blood-cells. The exact time of fetal death 
appears to vary in the case of individual mothers and, what is 
more remarkable, in the case of some embryos as contrasted 
with others in the same gestation. Thus dead and living . 
young may occupy neighboring sites in the same uterine 
horn. Embryos may succumb shortly after implantation, or 
again only shortly before term. 

Effect of Various Natural eel —Large numbers of 
females have been reared on various “‘pure’’ food regimens 
and bred shortly after the sixtieth day of life. Only those 
exhibiting a typical resorption were now employed to trace 
the distribution and abundance of the new food factor E in 
natural foods. Shortly after the incomplete or resorption 
gestation, a small amount of a single natural foodstuff was 
now added to the ration or fed separately from it, and the 
fate of the new gestation followed with similar care. In 
many instances a normal sized litter of vigorous young re- 
sulted. In others no alteration of the sterility was secured. 
We have thus charted the considerable and inconsiderable 
possession of E by common foods. 

Distribution of Vitamin E.—It is present but never highly 
concentrated in a great variety of animal tissues, muscles | 
fat, and viscera, included in the latter being pancreas, spleen , 
liver, heart, hypophysis, and placenta. One of the most 
remarkable things about the content of E in animal tissues 
is the fact that the vitamin is low in the viscera. It is lower 
in the liver than in the muscles. A daily feeding of half the 
total liver of rats reared on natural foods will not provoke 


RELATIONS BETWEEN FERTILITY AND NUTRITION 223 


fertility. There is failure also when the entire heart, spleen, 
brain, kidney, or testes are fed daily. The muscles and fat, 
on the other hand, while not a concentrated source of E, 
contain in their totality several times the minimal require- 
ment for a successful gestation. E is present, but to a small 
extent, in milk fat. Nine per cent of this, which is included 
in our basic ration, together with 15 per cent lard, fails to 
prevent sterility, though with lard absent 24 per cent suc- 
ceeds. Whole milk powder may constitute one-third of the 
ration by weight and sterility result. Yet when whole milk 
powder is the sole food, its fat content, 28 per cent, is suffi- 
ciently high to insure an adequate amount of E. There is 
definite evidence of a higher E content of milk given by 
cattle with access to fresh alfalfa pasturage. Cod-liver oil, 
though high in vitamins A and D, is notably lacking in E. 
Throughout the life of animals 9 per cent by weight of the 
ration may be constituted by cod-liver oil, a single drop of 
which daily is adequate for A requirements, and yet sterility 
results. In contrast with the paucity of E, even in its most 
abundant depots in animal tissues, is its concentration in the 
organs of certain plants, especially in seeds and green leaves. 
It can be demonstrated to be unhurt, after careful desicca- 
tion of such leaves (lettuce, alfalfa, pea, tea). Thus in a 
series of experiments, 1.5, 1, and finally 0.25 gm. daily of 
the lettuce leaf powder proved efficacious in cures. The con- 
tent of E is high in some cereals. We have found it in oats, 
corn, and especially wheat, where it is low in the endosperm, 
but concentrated in the embryo. The richness of wheat 
germ in E is extraordinary. We have found no other nat- 


224 LECTURES ON NUTRITION 


urally desiccated substance comparable to it in value; 250 
mg. daily evokes cures. In the case of both wheat germ and 
lettuce leaf, ether extraction of the carefully desiccated sub- 
stance removes E quantitatively and secures for us oils which 
are efficacious in daily, single drop (25 mg.) administrations. 
E is probably present in most commercial oils, so that when 
the latter constitute a high proportion of the diet, for in- 
stance, when fed as 15 per cent, displacing lard, fertility 
results. Such results have been secured with Wesson oil, 
cocoanut oil, olive oil. Cottonseed oil when hydrogenized 
constitutes the substance called Crisco. As is well known, 
it is practically devoid of vitamin A and has hence been 
frequently employed instead of lard in researches where an 
A-free diet was essential. Yet, when the fat content of our 
basic diet is represented by Crisco, fertility invariably re- 
sults, this being, in fact, a curative agent. Crisco, cottonseed 
oil, corn oil, olive, cocoanut, walnut, peanut, and flaxseed oils 
can all be fed daily in quantities five times the required 
minimum of wheat germ oil without restoring fertility. 

Proof of the Existence of Vitamin E in the Tissues of Am- 
mals Reared on Natural Foods and of Its Depletion in Those 
Reared on Synthetic Diets——We have completed a series of 
cannibal experiments. Sterile females reared on “pure” 
food were killed daily, and their tissues (liver, muscles, and 
fat) fed to other females reared in an identical fashion and 
likewise of proved sterilty. At the same time normal fe- 
males of proved fertility were similarly killed and fed to 
other sterile females reared on “pure” food. In all instances 
the tissues of rats reared on a natural food were able to 


RELATIONS BETWEEN FERTILITY AND NUTRITION 225 


cause fertility in their sterile sisters. Of even greater sig- 
nificance would seem the demonstration that in no instance 
could a cure be obtained by the administration of the same 
tissues from sterile females. 

The Survival of Fertility in Animals Shifted from a Diet 
Possessing Vitamin E to One Deprived of Ii.—Ili animals are 
reared on a diet of natural foodstuffs and, after their fertility 
is established, shifted to a pure food ration, they preserve 
their fertility for three or four months, and then lose it. 
Similarly, when sterile animals are cured with foods possess- 
ing the new vitamin, not only is the next gestation normal, 
but in some circumstances the next two or three gestations. 
The survival of normal fertility is roughly dependent on the 
amount of E in the curative diet. When by quantitative ex- 
periments we have determined the minimal dose of any 
“curative” food, that is, one capable of immediately re- 
storing fertility, we have been able to see the immediate 
loss of this fertility in the next gestation on the pure food 
regimen. 

Presence of Vitamin E in the Tissues of Normal Newborn 
Young.—Vitamin E is transferred from mother to offspring 
during intra-uterine life, for the tissue of newborn rats cures 
female dietary sterility. 

Proof of the Normal Use or Wastage of Vitamin E in the 
Usual Metabolic Processes of the Body.—Groups of females 
have been reared on a natural food and their fertility estab- 
lished by trial gestations, after which they were all shifted 
to our standard pure diet. Half of them were bred immedi- 


ately and in all instances were able to give birth to young in 
15 


226 LECTURES ON NUTRITION 


the next two succeeding pregnancies, the third uniformly 
failing. As soon as the advent of sterility was demonstrated, 
presumably by the exhaustion of E due to the drain of the 
repeated pregnancies, the other half of the animals were 
bred. These sisters were by this time likewise sterile. This 
half had been shifted to the pure food at the same time as 
had the first half of the group, but had been shielded from 
the drain of reproduction and especially placental function. 
Hence it seems clear that the body stores of vitamin E are 
employed in normal metabolic processes at approximately 
the same rate, whether or not we have the drain of gestation. 
An Excess of E Cannot Increase Fertility Beyond Normal 
Limits —The administration to sterile animals of foods or 
extracts of foods known to be twice to twenty times as rich 
in vitamin E as is required for the birth of living young does 
not increase size or weight of the litter, or in other ways im- 
prove the performance of the reproductive mechanism beyond 
normal limits. This is in consonance with what we know of 
the action of other vitamins, there being little or no reliable 
evidence of advantage from an abnormally high quota of 
them, yet absolute need of the minimal quota and, for com- 
plete normality, a need of what we can call the effective 
quota. | 
Efficacy of a Single Curative Dose of Vitamin E Admin- 
istered at the Beginning of Gestation.—Since the work which 
has previously been detailed showed a definite if transitory 
storage of E by the body, it seemed reasonable to suppose 
that a sufficiently high feeding of E on a single occasion 
(early in gestation) might suffice for that particular gesta- 


RELATIONS BETWEEN FERTILITY AND NUTRITION 227 


tion. It was, in fact, found that success resulted from a 
single administration of the same total amount represented 
in twenty-two days of separate daily dosage with the minimal 
effective amount of wheat germ oil. The minimal effective) 
daily dose was found to be about 25 mg., and a single ad-| 
ministration of 550 mg. of the oil led, in all tests, to the birth 
of living young. Furthermore, curative foods or extracts of 
those foods can be fed as late as the fifth or sixth day of 
pregnancy and save the situation. Finally, the vitamin in / 
the form of oil can be just as effectively administered paren- | 
terally (by subcutaneous or intraperitoneal injection) as by, 
mouth, _ : 
Physical and Chemical Characteristics of the New Sub- 
stance.—We come now to a consideration of the physical and 
chemical characteristics of the new substance. The vitamin 
may be called fat soluble, though its range of solubility is 
far greater than that of ordinary fats. While this range of 
solubility may really be due to the solubilities of impurities 
as yet associated with the vitamin, it is a fact that the most 
concentrated fractions yet obtained have been almost com- 
pletely miscible with solvents representing such a range as 
methyl alcohol, ethyl alcohol, ether, pentane, benzene, ace- 
tone, ethyl acetate, carbon disulfid, and so forth. The vita- 
min is almost insoluble in water, yet we have repeatedly en- 
countered its presence in water solutions. There is enough 
left in the water after precipitation of calcium soaps, for 
instance, to be extracted with ether and effect cures. The 
distribution ratio between water and ether is very large, for 
a few extractions with an equal volume of ether effect quan- 


228 LECTURES ON NUTRITION 


titative removal. This has been established by a large 
number of feedings of the non-saponifiable fraction, the resid- 
ual soap always failing to produce fertility. The solubility 
of E in such substances as alcohol and pentane shows a 
large temperature coefficient and is so much greater than 
some of the contaminating substances, the sterols, for ex- 
ample, as to permit separation of the vitamin from them. 

\/ Vitamin E is remarkably stable to heat, light, air, and 
‘many of the ordinary chemical reactions. As regards tem- 
perature, while the ashing of wheat germ completely de- 
stroyed the vitamin, yet heating of the germ to 170° C. so 
that it was greatly charred left the E unimpaired. Dis- 
tillation of wheat germ oil, or a fraction out of it, in super- 
heated steam at 180° C. for several hours has not destroyed 
it. Distillation in vacuo up to 233° C. has not, in fact, caused 
any lowering of the potency of the fractions so treated, nor 
have any physical changes like changes in solubility been 
detected. We have not encountered evidence that daylight 
affects E in wheat germ oil, but there would appear to be 
partial destruction by exposure in thin layers to a powerful 
quartz mercury lamp for one hour. As regards oxidation, 
exposure of wheat germ oil for as many as twelve hours to 
a stream of air washed with acid and alkali, and at 97° C., 
has not destroyed E. At normal temperatures the vitamin 
is remarkably stable to both acid and alkali and many chem- 
ical treatments. It dissolves unchanged, for instance, in sat- 
urated alcoholic hydrogen chlorid. We have hydrogenized 
wheat germ oil in the presence of palladium at 75° C., and no 
injury to the vitamin resulted. Further, alcoholic extracts 


RELATIONS BETWEEN FERTILITY AND NUTRITION 229 


of Crisco, a hydrogenization product of cottonseed oil, are 
always fairly rich in the vitamin. We have treated the 
germ oil with both 20 per cent hydrochloric acid and one- 
tenth normal acid for twenty hours at room temperatures 
without destruction of the vitamin. It is not destroyed by 
concentrated sulfuric acid. It resists the action of boiling 20 
per cent alcoholic potassium hydrate, though partial destruc- 
tion would appear to occur on very prolonged hot saponi- 
fication. The saponification with 20 per cent alcoholic potas- 
sium hydrate can be carried out at 30° C. without great loss 
of the vitamin which goes into the non-saponifiable quota, 
5 per cent of the oil, so that by this step alone a notable con- 
centration of E is always attained. ‘The non-saponifiable 
quota is, in turn, chiefly (73 per cent) sitosterol, which is 
largely insoluble in pentane in the cold, an excellent solvent 
for vitamin E, which, together with pigments and other 
materials, can thus be washed out of the sterols, leaving them 
white. The sterols are inactive. If the orange-red viscous 
oil obtained from the pentane is treated with methyl al- 
cohol, more extraneous material is removed, and the vita- 
min goes into the alcohol portions which now can be mixed 
with petroleum ether or diluted to 90 per cent methyl alco- 
hol, allowing an immediate separation into two layers, the 
petroleum ether invariably securing more of the vitamin, 
in fact, all of it, if the distribution be done with successive 
fresh portions of the petroleum ether. Further purifications 
can now be carried out both with digitonin, boiling methy- 
alcohol, and finally, distillations in vacuo; yet the concentra- 
tion effected, of course, does not relatively compare with 


230 LECTURES ON NUTRITION 


that effected with the first three steps of the procedure just 
outlined. The final yellow viscous oil does not develop 
crystals on long standing. It contains only a trace of ash and 
no nitrogen, sulphur, phosphorus, or halogen. It is remark- 
ably potent. When 5 mg. are fed or injected under the skin 
of a female of proved sterility at the inception of a new 
gestation, normal litters of vigorous young are born and 
have been reared to adolescence. Sister control rats invari- 
ably continue sterile. Furthermore, the daily administra- 
tion of only 0.3 mg. of this substance throughout the life 
of the male results in the retention of complete normality 
when animals are reared and held on pure foods, a normality 
proved by the weight and histologic condition of the testis 
and by weekly functional tests throughout a year, and con- 
trolled by the invariable development of sterility at the 
end of three months in litter-mate brothers held on the iden- 
tical ration save for omission of the trace of vitamin. 


RELATIONS BETWEEN FERTILITY AND NUTRITION 231 


OUTLINE OF FRACTIONATION OF 6 KG. OF WHEAT GERM 


Six kilograms wheat germ. 


Extracted with U. S. P. 
Ether in Soxhlet. 


Ether extract. Active. 
Yield: 600 gm. 10 per cent. 


| 
Saponified in the cold with 
20 per cent alcoholic po- 
tassium hydrate. 


Nonsaponifiable matter (N.S. M.). 
Yield: 5 per cent. Contains all 
the active material. 


| 
Crystallized from cold pen- 
tane. 


| 

Pentane-soluble red oil. Yield: 33 
per cent of the N.S. M. Con- 
tains all active material. 


| 
Extracted with hot methyl 
alcohol. 


| 
Hot methyl alcohol solution. 
Active. 


| 
Crystallized from cold methyl 
alcohol. 


| 
Cold methyl alcohol solution. 
Active. 


| 
Distributed between dilute 
methyl alcohol and _ pe- 
troleum ether. 


Ether insoluble residue. 
Inactive. 


Soaps and glycerol. 
Inactive. 


Pentane-insoluble solids. 
Sitosterol. Yield: 66 per cent of 
N.S. M. Inactive. 


| 
Methyl alcohol insoluble residue. 
Inactive. Yield: 3 per cent of 
N.S. M. 


Precipitate from cold methyl Be 
cohol. Almost entirely inac- 
tive. Yield: 10 per cent of 
N.S. M. 


232 LECTURES ON NUTRITION 


Petroleum ether soluble. Active. 
Yield: 13 per cent of N.S. M. 


Sterols precipitated by digi- 
tonin. 


Sterol-free orange oil. Yield: 2 
to 3 gm. Active. 


Pe aN epee 
Refluxed in hot 20 per cent 
alcoholic potassium hydrate. 


N. S. M. contains all the active 
material. 


Sterols again precipitated by 
digitonin. 


Sterol-free oil. Active. 


| 
Treated with boiling methyl 
alcohol. 


Orange solution in methyl alcohol. 
Active. Yield: 700 to 1000 
mg. (proved active in single 
doses of 5 to 10 mg.). 


Distilled in vacuo. 


Fraction I 


Fraction II 


, | 
Dilute methyl alcohol soluble. In- 
active. Yield: 4 per cent of 
N.S. M. 


Sterols from digitonide. 
Inactive. 


Fatty acids. Inactive. 


Sterols. Inactive. 


Residue insoluble in hot methyl 
alcohol. Inactive. Yield: 50 
to 100 mg. 


| 
Fraction III 


Up to 200° C.at 0.8mm. 200° to 233° C. at 0.5 Residue above 233° C, 


Low acidity. mm. 
33 per cent of total. 


Highly active. 


Highly active. 
33 per cent of total. 


27 per cent of total. 


RELATIONS BETWEEN FERTILITY AND NUTRITION 233 


BIBLIOGRAPHY 


1. Evans, H. M.: Unique Dietary Needs for Lactation, Science, 1924, 
lx, 20. 


2. Evans, H. M., and Bishop, Katherine S.: (Estrus and Ovulation in the 
Rat from a Large Group of Animals on a Standard Diet, Anat. 
Rec., 1922, xxiii, 17-18. 

On an Invariable and Characteristic Disturbance of Reproductive 
Function in Animals Reared on a Diet Poor in Fat-soluble Vitamin 
A, Anat. Rec., 1922, xxiii, 17. 

On the Relations Between Fertility and Nutrition. I. The Ovulation 
Rhythm in the Rat on a Standard Nutritional Régime, Jour. Met. 
Res., 1922, i, 319. 

II. The Ovulation Rhythm in the Rat on Inadequate Nutritional 
Regimens, Jour. Met. Res., 1922, i, 355. 

III. The Normal Reproductive Performance of the Rat, Jour. Met. 
Res., 1923, iii, 201. 

IV. The Production of Sterility with Nutritional Régimes Adequate 
for Growth and Its Cure with Other Food-stuffs, Jour. Met. 
Res., 1923, ili, 233. 

On the Existence of a Hitherto Unrecognized Dietary Factor 
Essential for Reproduction, Science, 1922, ivi, 650. 

Existence of a Hitherto Unrecognized Dietary Factor Essential for 
Reproduction, Jour. Am. Med. Assn., 1923, Ixxxi, 889. 

The Cause of Reproductive Upset in Dietary Deficiencies Due to 
Lack of Vitamin A, Anat. Rec., 1923, xxv, 129. 

Stability and Solubilities of the Food Substance or Vitamin X 
Required for Reproduction, Anat. Rec., 1924, xxvii, 203. 

Proof of the Power of the Body to Store the Substance X Required 
for Reproduction, Anat. Rec., 1924, xxvii, 204. 


3. Evans, H. M., and Burr, G. O.: Preliminary Steps in the Isolation 
and Concentration of Vitamin X, Anat. Rec., 1924, xxvii, 203. 
Distribution of Vitamin X in Natural Foods, Anat. Rec., 1925, xxix, 
356. 


4, Hopkins, F. G.: The Analyst and the Medical Man, Analyst, 1906» 
xxxi, 385, 


5. KG6nigstein, H.: Die Verainderungen der Genitalschleimhaut wahrend 
der Graviditit und Brunst bei einigen Nagern, Arch. f. Physiol., 
1907, cxix, 533. 


6. Lataste, F.: Transformation périodique de l’epithélium du vagin des 
rongeurs (rhythme vaginal), Mem. d. Soc. d. biol., 1892, xliv, 765. 
Rhythme vaginal des mammiféres, Mem.d.Soc.d. biol., 1893, xlv, 135. 


234 LECTURES ON NUTRITION 


7. Long, J. A., and Evans, H. M.: The Cstrous Cycle in the Rat and 
Its Associated Phenomena, University of California Memoirs, 
University of California Press, Berkeley, 1922, vi. 

8. McCollum, E. V.: The Newer Knowledge of Nutrition, 2d ed., New 
York, Macmillan, 1922. 

9. Morau, H.: Des transformations epitheliales de la muqueuse du vagin 
de quelques rongeurs, Jour. Anat. et d.1. Physiol., 1889, xxv, 275. 

10. Osborne, T. B., and Mendel, L. B.: Formula for Our ‘Standard Basic 
Ration”’ given in a letter dated February 10, 1921. 

11. Retterer, E.: Sur les modifications de la muqueuse uterine 4 l’epoque 
du rut, Mem. e. Compt. rend. d. Soc. d. biol., 1892, xliv, 637. 

12. Stockard, C. R., and Papanicolaou, G. N.: The Existence of a Typical 
(Estrous Cycle in the Guinea-pig with a Study of Its Histological 
and Physiological Changes, Am. Jour. Anat., 1917, xxii, 225. 

13. Wolbach, S. B., and Howe, P. R.: The Epithelial Tissues in Experi- 
mental Xerophthalmia, Proc. Soc. Exper. Biol. and Med., 1925, 
xxii, 402. 


INDEX 


ABEL and Takamine, 63 
Accumulator function of muscle, 124 
Acid, lactic, 112 
carbohydrate origin of, 114 
fate of, in recovery, 119 
in man, 126 
Activity, muscular, and metabolism, 
24 
Age a factor in metabolism, 23 
Alcohol and basal metabolism, 45 
Anabolism, 18, 20 
Analogous metabolism in other cells, 
116 | 
Anrep and Drummond, 160 
Antiglyoxalase, 118 
Apprehension and metabolism, 45 
Azuma and Hartree, 115 


BACTERIA, intestinal, increase due to 
deficiency of vitamin A, 148 
Barcroft, 117 
Barlow, 181 
Barnett, Zucker, and Johnson, 191 
and Pappenheimer, 193 
Barr and McCann, 103 
Cecil, and DuBois, 88 
Himwick, and Loebel, 115 
' Basal katabolism, 20 
metabolism. See Metabolism, ba- 
sal: 
Beach, 154 
Bedson and Zilva, 149 
Benedict, 17, 30, 32, 36, 41, 77, 81, 
92, 93 
and Sugiura, 154 


Beri-beri due to lack of vitamin B, 
155 
pathology of, 164 
Berzelius, 59 
Bethke, Steenbock, and Nelson, 192 
Bezssonov, 175 
Biedl, 60 
Billroth, 60 
Bios, 138 
and growth of yeast, 169 
Biostearin, 152, 153 
Bishop and Evans, 146, 201, 202 
Black and Steenbock, 199 
Blegvad, 142 
Bloch, 140, 141 
and Mori, 141 
Blood-platelets, decrease in, from 
vitamin A deficiency, 148 
Body position, influence of, on met- 
abolism, 32 
Boothby, 81 
and Sandiford, 104 
Bosanyi, 191, 192 
Boutwell and Steenbock, 152 
and Kent, 151 
Boyenval, 161 
Breakfast, permissible, prior to met- 
abolism tests, 35 
Buchholz, 197 
Burge, 161 
Burhans and Gerstenberger, 177 
Burr, 209 


CALoRIE, 17 
Calorimeter, emission, 39 


235 


236 


Calorimetry, direct, 27 
measurement of intensity of 
combustion by, 25 
indirect, 27 
Campbell and LaMer, 177 
Carbohydrate and fat oxidation, 
and relation to ketosis, 95 
metabolism of, 71 
and muscular activity, 109 
origin of lactic acid, 114 
protein, and fat, proportions in 
which metabolized in disease, 77 
Carpenter, 46, 47 
Cecil, Barr, and DuBois, 88 
Cessna, Nelson, and Fulmer, 171, 
172 
Chambers, 74 
Cod-liver oil in rickets, 183, 189, 190 
Cohen and Mendel, 176 
Coleman and DuBois, 101 
and Shaffer, 100, 101 
Coward and Drummond, 149 
Cowgill, 160, 163 
and Mendel, 161 
Cramer, 105, 142, 147, 148, 149 
Drew, and Mottram, 148, 149, 167 


DAKIN and Dudley, 118 

Darbey, 190 

Davis, 151 
and McCollum, 140 

Deas, 172 

DeGouvea, 142 

Deuel, 74 

Diabetes and fat, 97 
respiratory quotient in, 85 

Diets, different, mechanical effici- 
ency on, 110 

Differences in individuals, 22 

Direct calorimetry, 27 

Drew, Cramer, and Mottram, 148, 
149, 167 

Dreyer, 52 


INDEX 


Drummond and Anrep, 160 
and Coward, 149 

DuBois, 36, 44, 52, 53, 63, 77 
and Coleman, 101 
Cecil, and Barr, 88 

Dudley and Dakin, 118 
and Marrian, 115 

Dutcher, 156, 161, 162, 174 
and Kennedy, 164 


Eppy and Kohlman, 174 
Kerr, and Williams, 172 
Efficiency of recovery, 121 
Eijkman, 138 
Embden, 112, 113 
Emission calorimeter, 39 
Emmett and Luros, 152 
and Peacock, 154 
Energy, metabolism of, 20 
Environmental temperature and ba- 
sal metabolism, 24, 37 
Evans, 209 
and Bishop, 146, 201, 202 
Exercise and recovery, respiratory 
quotient of, 132 
oxygen requirement of, 130 
Eye symptoms from lack of vitamin 
A, 139 


Factors involved in metabolism, 
20 
Falta and Noeggerath, 139 
Fasting in metabolism, 92 
Fat and carbohydrate oxidation and 
relation to ketosis, 95 
and diabetes, 97 
metabolism of, 71 
nutrient qualities of, 140 
protein, and carbohydrate, pro- 
portions in which metabolized 
in disease, 77 
Fear and metabolism, 45 


INDEX 


Fertility and nutrition, relation be- 
tween, 209, 212 
cannot be increased beyond nor- 
mal limits by excess of vitamin 
E, 226 
survival of, in animals shifted 
from diet possessing vitamin E 
to one deprived of it, 225 
Fever and basal metabolism, 24, 44 
Fick and Wislicenus, 87 
Findlay, 161, 162, 168 
Fingerling, 140 
Finley and Parker, 73 
Fisher, 71, 81 
Fletcher, 112 
and Hopkins, 112, 119 
Folin, 71 
Food hormones, 137 
ingestion, 20 
effect on metabolism, 33 
Foster and Moyle, 114 
and Woodrow, 115, 116, 117, 119 
Frohlich and Holst, 173 
Fulmer, Nelson, and Cessna, 171, 
Li 
Funk, 156, 162 
Fiirst, 173 
Furusawa, 133, 134, 135 


GASEOUS metabolism, 20 
Gerstenberger and Burhaws, 177 
Gestation, efficacy of single cura- 
tive dose of vitamin E admin- 
istered at beginning of, 226 
resorption, 220 
Giardia intestinalis in vitamin A 
deficiency, 148 
Glisson, 181 
Glucose, 99 
in nervousness, 73 
Glyoxalase, 118 
Grafe, 57 
Green, 162 


237 


Grijns, 138 
Grouven, 35 
Guy, 190 


HARDEN and Young, 113 
Harris, 54 
Hart, 174 
Hartree and Azuma, 115 
Heart failure, metabolism in, 104 
Heat loss, 25, 27 
and heat production, 25 
production, 25 
and basal metabolism, 28 
and heat loss, 25 
effect of proteins and deriva- 
tives on, 69 
relation to surface area, 66 
variability of, 65 
Height and metabolism, 22 
Hess, 200 
and Weinstock, 200 
Unger, and Pappenheimer, 177 
Hetzel, Long, and Lupton, 115 
Hikan, 145 
Hill, 74, 87, 88 
and Webster, 199 
Himwich, Loebel, and Barr, 115 
Histamin, relation of, to vitamin B, 
160 
Hofmeister, 72 
Hojer, 178 
Holst and Frohlich, 173 
Hopkins, 189, 210 
and Fletcher, 112, 119 
and Wingfield, 116, 119 
Hormones, food, 137 


Howe and Wolbach, 217 


Howland and Kramer, 186, 194, 195 
and Marriott, 193 

Huldschinsky, 198 

Hulshoff-Pol, 138 

Hume, 198 
and Smith, 198 


238 


Hun and Sanger, 105 

Hyperthyroidism, metabolism 
104 

Hypoglycemia, 73, 74 


in, 


INDIRECT calorimetry, 27 
Individuals, differences in, 22 
Infantile tetany, causes, 193 
Infection and basal metabolism, 100 
Ingestion of food, 20 
effect on metabolism, 33 

Intestinal bacteria increased from 

vitamin A deficiency, 148 
Iwabuchi, 177, 178 


JoHANsSON, 37, 38 
Johnson, Barnett, and Zucker, 191 


Karr, 163 
Katabolism, 18, 20 
basal, 20 
Kaye and Robison, 113 
Kendall, 63 
Kennedy and Dutcher, 164 
and Palmer, 151 
Kent, Steenbock, and Boutwell, 151 
Kerr, Eddy, and Williams, 172 
Ketosis, relation of carbohydrate 
and fat oxidation to, 95 
symptoms of, 97 
Klemperer, 91 
Kocher, 87, 101 
Kohman and Eddy, 174 
K6nigstein, 213 
Koskowski, 161 
Kramer and Howland, 186, 194, 195 
Krogh, 81 
and Lindhard, 110, 111 


LACTACIDOGEN, 113 

Lactic acid, 112 
carbohydrate origin of, 114 
fate of, in recovery, 119 
in man, 126 


INDEX 


Ladd and Richardson, 81, 94 
Lambert and Yudkin, 143 
LaMer and Campbell, 177 
Landergren, 91, 101 
Lataste, 213 
Lavoisier, 65, 209 

and Séguin, 33 
Lefévre, 30, 41, 42 
Levene, 63, 158, 159 

and Richardson, 88, 105 

and van der Hoeven, 157 
Liebig, 59, 60, 209 
Light, effect of, in prevention and 

cure of rickets, 197 
Lind, 179 
Lindhard and Krogh, 110, 111 
Ling and Wierzuchowski, 72 
Liotta, 178 
Livingstone, 138, 139 
Loebel, Himwich, and Barr, 115 
Loewy, 37 
Long and Lupton, 126 

and Hetzel, 115 
Lupton and Long, 126 
and Hetzel, 115 

Luros and Emmett, 152 
Lusk, 50, 57, 80, 85, 94 
Lymphopenia, 149 

from deficiency of vitamin B, 167 


MacDona.p, 171 
Magnus-Levy, 63 
Man, lactic acid in, 126 
Marasmus, 167 . 
Marrian and Dudley, 115 
Marriott and Howland, 193 
Mason and Richardson, 97 
McCann and Barr, 103 
McCarrison, 155, 164, 165, 166, 167, 
177 
McCollum, 209, 219 
and Davis, 140 
and Simmonds, 141, 143, 158, 160 
and Souza, 170 


INDEX 


McCollum, Park, Shipley, Aa Sim- 
monds, 184, 186 
McGowan and McNeil, 142 
McNeil and McGowan, 142 
Mechanical efficiency on ee 
diets, 110 
Mellanby, 182, 183, 189 
Mendel, 60 
and Cohen, 176 
and Cowgill, 161 
and Osborne, 142, 151, 152 
Mental attitude and metabolism, 44 
Metabolism, 17, 62 
analogous, in other cells, 116 
basal, 63 | 
age a factor in, 23 
alcohol and, 45 
and environmental 
ture, 24 
and fever, 24 
and infection, 100 
and nutrition, 24 
and sleep, 24 
apprehension and fear, effects, 
45 
comparison of, between indi- 
viduals, 50 
current standards of reference, 
52 
differences in individuals and, 
22 
DuBois standard of reference, 
54 
effect of food ingestion, 33 
of muscular work or repose, 
29 
preceding muscular activity, 
oe 
environmental temperature and, 
37 
factors involved in, 20 
fever and, 44 
heat production and heat loss 
in, 25 


tempera- 


Metabolism, basal, influence of body 
position on, 32 
ingestion of food and, 20 
in hyperthyroidism, 104 
in tuberculosis, 103 
in typhoid fever, 100-103 
measurement and _ significance 
ord 7 
measurements, conditions speci- 
fied for, 47 
mental attitude and psychic re- 
pose in, 44 
minor muscularmovementsand, 
30 
muscular activity and, 23 
permissible breakfast prior to 
test, 35 
Pirquet’s pelidisi in measure- 
ment, 55 
psychic disturbances and, 24 
reproducible experimental con- 
ditions, 28 
seasonal variability, 49 
sleep and, 45 
standards of, 49 
state of nutrition and, 56 
surface area law and, 51 
test in water-bath, 41, 42 
carbohydrate, and muscular ac- 
tivity, 109 
definition, 17 
gaseous, 20 
in heart failure, 104 
in renal disease, 104 
nitrogenous, 19 
of energy, 19 
of fat and carbohydrate, 71 
problems of, 59 
Meyerhof, 74, 112, 113, 114, 119, 
120, 121 
Milk, vitamin C, content of, 173 
Miller, 172 
Miura and Zilva, 188 
Morau, 213 


240 


Morel, 177 
Mori, 139, 143, 144, 146 
and Bloch, 141 
Morikawa, 177 
Motttam, Cramer, and Drew, 148, 
149, 167 
Moyle and Foster, 114 
Miiller, 34 
Muscle, accumulator function of, 124 
Muscular activity and carbohydrate 
metabolism, 109 
and metabolism, 23 
preceding metabolism test, ef- 
fect of, 33 
movements, minor, and metabo- 
lism, 30 
work or repose, effects on met- 
abolism, 29 
Myers and Voegtlein, 160 


NEtson, Bethke, and Steenbock, 192 
Fulmer, and Cessna, 171, 172 
Nephritis, metabolism in, 104 
Nervousness, glucose in, 73 
Neuberg, 73 
Neumann, 197 
Nitrogen minimum, Thomas on, 91 
Nitrogenous metabolism, 19 
Noeggerath and Falta, 139 
Nutrition and basal metabolism, 24, 
56 
and fertility, relation between, 
209, 212 
relation to reproduction, 211 


OBEsITY, 98 
(Estrus and ovulation, effect of 
vitamins A and B on, 215 
cycle disturbed by deficiency of 
vitamin A, 146 
vaginal changes during, 212 
Ophthalmia from lack of vitamin A, 
139 
of dietary origin, 143 


INDEX 


Osborne and Mendel, 140, 142, 151, 
152 
and Wakeman, 157, 158 
Ovulation and cestrus, effects of 
vitamins A and B on, 215 
Oxidative quotient, 121 
Oxygen requirement of exercise, 
130 


Paum, 197 
Palmer and Kennedy, 151 
Pancreatic control, 115 
Papanicolaou and Stockard, 213 
Pappenheimer, 191 
and Sherman, 184, 185 
Hess, and Unger, 177 
Zucker, and Barnett, 193 
Park, Shipley, McCollum, and Sim- 
monds, 184, 186 
Parker and Finley, 73 
Pasteur, 170 
Peacock and Emmett, 154 
Petersen, 196 
Physiology of reproduction of rats, 
211 
Pirquet’s pelidisi 
measurement, 55 
Placental sign, 201 
Polyneuritis, 164 
from deficiency of vitamin B, 155, 
156 
Pritchard and Webster, 169 
Proportions in which protein, fat, 
and carbohydrate are metabolized 
in disease, 77 
Proteins and derivatives, effect of, 
on heat production, 69 
fat and carbohydrate, propor- 
tions in which, metabolized in 
disease, 77 
Psychic disturbances and metabo- 
lism, 24 
repose and metabolism, 44 


in metabolism 


INDEX 


QUOTIENT, oxidative, 121 
respiratory. See Respiratory quo- 
tient. 


RACZYNSEI, 197 
Radium in rickets, 200 
Rapport, 63 
and Weiss, 69 
Rats, physiology of reproduction of, 
211 
Recovery and exercise, respiratory 
quotient of, 132 
process, 122 
in man, 125 
Renal disease, metabolism in, 104 
Repose or muscular work, effects on 
metabolism, 29 
Reproduction and vitamins, 201 
of rats, physiology of, 211 
relation to nutrition, 211 
Resorption gestation, 220 
Respiratory quotient, 79, 110 
after severe muscular exercise, 


129 
during and after muscular ex- 
ercise, 128 


in diabetes, 85 
of exercise and recovery, 132 
use of, 130 
Retterer, 213 
Richardson, 77 
and Ladd, 81, 94 
and Levene, 88, 105 
and Mason, 97 
Rickets, 180 
cod-liver oil in, 189, 190 
description, 181 
effect of light in prevention and 
cure, 197 
etiology, 184 
experimental, 182 
radium in, 200 
Ringer, 73 


241 


Robison and Kaye, 113 

Rosenfeld, 95 

Rubner, 33, 34, 52, 60, 65, 66, 67, 68, 
69, 70, 71, 77, 91, 209 

Rudinger, 105 


SALIVARY glands, dysfunction from 
lack of vitamin A, 146 
Sandiford and Boothby, 104 
Sanger and Hun, 105 
Schaumann, 138 
Scurvy, anatomic lesions in, 176 
from deficiency of vitamin C, 
173 
Seasonal variability in basal met- 
abolism, 49 
Séguin and Lavoisier, 33 
Seidell, 159 
Shaffer, 73 
and Coleman, 100, 101 
and Woodyatt, 95, 96 
Sherman, 217 
and Pappenheimer, 184, 185 
Shinza, 157 
Shipley, Park, McCollum, and Sim- 
monds, 184, 186 
Simmonds and McCollum, 141, 143, 
158, 160 
Park, Shipley, and McCollum, 
184, 186 
Sivén, 91 
Sizi, indication, 22 
Skin, temperature of, 27 
at different parts of body, 25 
Slater, 119 
Sleep and basal metabolism, 24, 45 
Smith, 178 
and Hume, 198 
Souza and McCollum, 170 
Starling and Verney, 124 
Starvation in metabolism of pro- 
tein, fat, and carbohydrate, 92 
Steenbock, 150 


242 : INDEX 


Steenbock and Black, 199 
and Boutwell, 152 
Bethke, and Nelson, 192 
Boutwell, and Kent, 151 
Stehle, 161 
Stephenson and Whetham, 117 
Stepp, 139, 140 
Sterility disease, 218 
effect of various natural food- 
stuffs on, 222 
Stockard and Papanicolaou, 213 
Sugiura and Benedict, 154 
Sure, 202 ; 
Surface area and metabolism, 22, 23 
law and basal metabolism, 51 
relation to heat production, 66 
Suzuki, 152 


TAKAHASHI, 152 
Takamine and Abel, 63 
Temperature, environmental, and 
basal metabolism, 24, 37 
of skin, 27 
at different parts of body, 25 
Tetany, infantile, causes, 193 
Thomas, 87, 91, 94 
on nitrogen minimum, 91 
Thrombopenia, 149 
Tuberculosis, metabolism in, 103 
Typhoid, metabolism in, 100-103 


UNDERNUTRITION in metabolism of 
protein, fat, and carbohydrates, 
92, 100 

Unger, Hess, and Pappenheimer,177 


VAGINAL changes during cestrual 
cycle, 212 

van der Hoeven and Levene, 157 

VanSlyke, 71 

Vedder, 155, 162 


Verney and Starling, 124 
Vitamins, 17 
A, 138 


and B, effect on ovulation and 
oestrus, 215 

diseases due to deficiency of, 142 

lesions in digestive tracts from 
deficiency of, 147 

occurrence of, 150 

origin, 149 

properties, 151 

requirements of different species 
for, 154 


and reproduction, 201 
B, 155 


nature of action of, 162 
occurrence of, 155 

relation of histamin to, 160 
studies on isolation of, 156 


Cy.173 


occurrence of, 174 


D, 180 
E, 212, 218 


distribution of, 222 

efficacy of single curvative 
dose administered at begin- 
ning of gestation, 226 

excess of, cannot increase fer- 
tility beyond normal limits, 
226 

fat-soluble, 227 


physical and chemical charac- 


teristics of, 227 
presence of, in tissues of normal 
newborn young, 225 
proof of existence of, in tissues 
of animals reared on nat- 
ural foods and depletion in 
those reared on synthetic 
diets, 224 
of use or wastage of, in met- 
abolism, 225 


present knowledge, 137 
Voegtlin and Myers, 160 


INDEX 243 


Voidt, 57, 59, 60, 62, 81 Wolbach and Howe, 217 
von Voit, 209 Woodrow and Foster, 115, 116, 117, 
119 


Woodyatt and Shaffer, 95, 96 
WAKEMAN and Osborne, 157, 158 


Walshe, 155 

Warbury, 117, 118 x, 202, 210 

Wason, 143 Xerophthalmia from deficiency of 
Webster and Hill, 199 vitamin A, 141, 145 


and Pritchard, 169 
Weight and metabolism, 22 


Weinstock and Hess, 200 YEAST, 157, 163 

Weiss, 60 growth of, and bios, 169 
and Rapport, 69 Young and Harden, 113 

Wells, 177. Yudkin and Lambert, 143 


Whetham and Stephenson, 117 
Wildiers, 170 


Wierzuchowski and Ling, 72 ZILVA, 176 
Williams, 156, 170 and Bedson, 149 
Eddy, and Kerr, 172 and Miura, 188 
Winfield and Hopkins, 116, 119 Zucker, Johnson, and Barnett, 191 
Wishart, 71 Pappenheimer, and Barnett, 193 


Wislicenus and Fick, 87 Zuntz, 33, 34 


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